mRNA splice variant of the doublecortin-like kinase gene and its use in cancer diagnosis and therapy

ABSTRACT

The present invention relates to novel nucleic acid and protein molecules and their use in neuroblastoma therapy and diagnosis.

FIELD OF THE INVENTION

The present invention relates to a novel doublecortin like protein (DCL)and a novel mRNA splice variant encoding it. Provided are mouse andhuman nucleic acid sequences (RNA and DNA) encoding the novel DCLprotein, as well as the mouse and human protein itself and variousnucleic acid fragments and variants suitable for therapeutic anddiagnostic applications. The invention further relates to methods formodulating DCL protein levels in cancer therapy, especiallyneuroblastoma therapy, and to diagnostic methods and diagnostic kits.

BACKGROUND OF THE INVENTION

As the most common solid tumor in children, neuroblastoma accounts for8-10% of all cancers in children (for review see Lee et al., 2003, Urol.Clin. N. Am. 30, 881-890). Annual incidence ranges from 10 to 15 per100,000 infants, according to population based screening conducted inCanada, Germany and Japan. Neuroblastoma is a heterogeneous disease,with 40% diagnosed in children under 1 year of age who have a very goodprognosis, and the rest in older children and young adults who have apoor prognosis despite advanced medical and surgical management. Acommon treatment for intermediate- and high-risk patients ischemotherapy followed by surgical resection. However, completeeradication of neuroblastoma cells is seldom achieved. Consequently, themajority of these patients undergo relapse, which is often resistant toconventional treatment and rapidly overwhelming. Thus, after inductionof the apparent remission by the first-line therapy, new therapeuticstrategies are needed to completely eradicate the small number ofsurviving cells, to prevent relapse (Lee et al., 2003, supra).

Brain development requires the co-ordinated and precise patterning ofcell division, migration and differentiation of neuroblasts (Noctor etal., 2001, Nature 409, 714-720; Noctor et al., 2004, Nat. Neuroscience7, 136-144). A key event in both these processes is the (re)organizationand (de)stabilization of the cytoskeleton, which is comprised ofmicrotubules and microtubule-associated proteins (MAPs). A carefullyorchestrated interaction of microtubules with several MAPs is requiredbefore neuronal migration can occur (reviewed in Feng and Walsh, 2001,Nat. Rev. Neurosci. 2, 408-416). Although the factors involved inneuronal migration are well established, relatively little is knownabout the genes that control earlier processes, like mitosis andneuroblast proliferation. Such factors very likely involve dynamicregulation of the microtubular and cytoskeletal elements as well (Haydaret al., 2003, Proc. Natl. Acad. Sci. 100, 2890-2895; Kaltschmidt et al.,2000, Nat. Cell Biol. 2, 7-12; Knoblich, 2001, Nat. Rev. Mol. Cell Biol.2, 11-20).

Recently, several genes involved in cytoskeleton reorganization havebeen identified that, when disrupted or mutated, cause neuronalmigration disorders (reviewed in Feng and Walsh, 2001, supra). One ofthese genes is doublecortin (DCX) that encodes a 365 AA protein criticalfor migration of newborn cortical neurons (see WO99/27089). In the humanand rodent genome, a related gene, called doublecortin-like kinase(DCLK), is present that has substantial sequence identity with the DCXgene. The human DCLK gene spans more than 250 kb and is subject toextensive alternative splicing, generating multiple transcripts encodingdifferent proteins (Matsumoto et al., 1999, Genomics 56, 179-183). Oneof the main transcripts, DCLK-long, encodes a DCX domain fused to akinase-like domain that has amino acid homology with members of theCa++/Calmodulin dependent protein kinase (CaMK) family. Anothertranscript, DCLK-short, is mainly expressed in adult brain, lacks theDCX domain and encodes a kinase with CaMK-like properties (Engels etal., 1999, Brain Res. 835, 365-368; Engels et al., 2004, Brain Res. 120,103-114; Omori et al., 1998, J. Hum. Genet. 43, 169-177; Vreugdenhil etal., 2001, Brain Res. Mol. Brain Res. 94, 67-74). Recent studies suggestimportant roles for the DCLK gene in calcium-dependent neuronalplasticity and neurodegeneration (Burgess and Reiner, 2001, J. Biol.Chem. 276, 36397-36403; Kruidering et al., 2001, J. Biol. Chem. 276,38417-38425). DCLK-long is expressed during early development (Omori etal, 1998, supra) and like DCX, is capable of microtubule polymerization(Lin et al., 2000, J. Neurosci. 20, 9152-9161). However, the preciserole of the DCLK gene in development of the nervous system is unknown.

Various alternative splice-variants of DCLK have been described and twoof these have been found to be differentially expressed and to havedifferent kinase activities (Burgess and Reiner 2002, J. Biol. Chem.277, 17696-17705). The present inventors cloned and functionallycharacterized a novel splice variant of the DCLK gene, referred to asdoublecortin-like (DCL) herein, and have shown that DCL is acytoskeleton gene which is associated with mitotic spindles of dividingneuroblasts. In addition, the present inventors have devised novelmethods for cancer therapy and diagnosis, especially for neuroblastomatherapy and diagnosis.

Recently, new approaches for treatment of neuroblastoma have beenpublished, involving the use of antisense oligonucleotides targeting twodifferent oncogenes (Pagnan et al., 2000, J. Natl. Cancer Inst. Vol 92,253-261; Brignole et al. 2003, Cancer Lett. 197, 231-235; Burkhart etal., 2003, J. Natl. Cancer Inst. 95, 1394-1403). The first approach wasdirected against the c-Myb oncogene (Pagnan et al., 2000, supra). C-Mybgene expression has been reported in several solid tumors of differentembryonic origins, including neuroblastoma, where it is linked to cellproliferation and differentiation. It was shown that a phosphorothioateoligodeoxy-nucleotide complementary to codons 2-9 of human c-Myb mRNAinhibited growth of neuroblastoma cells in vitro. Its inhibitory effectwas greatest when it was delivered to the cells in sterically stabilizedliposomes coated with a monoclonal antibody (mAb) specific for theneuroectoderma antigen disialoganglioside GD₂ (Pagnan et al., 2000,supra). Although pharmaco-kinetic and biodistribution studies afterintravenous injection of anti-GD₂-targeted liposomes have been performed(Brignole et al., 2003, supra), the effect in an in vivo neuroblastomamodel has not been shown so far. Potential toxic side-effects of a c-Mybantisense oligonucleotide should also be considered, since the c-Mybprotein plays a fundamental role in the proliferation of normal cellsand it has already been shown that a c-Myb antisense oligonucleotideinhibits normal human hematopoiesis in vitro (Gewirtz and Calabretta,1988, Science 242, 1303-1306).

Another antisense approach was directed against the MYCN(N-myc) oncogene(Burkhart et al., 2003, supra). Amplification of the MYCN gene occurs inonly 25 to 30% of neuroblastomas, but is associated with advanced-stagedisease, rapid tumor progression and a survival rate of less than 15%.The effect of a phosphorothioate oligodeoxynucleotide complementary tothe first five codons of human MYCN mRNA was tested in vivo in a murinemodel of neuroblastoma. It was shown that continuous delivery of theoligonucleotide for 6 weeks via a subcutaneously implanted microosmoticpump could decrease tumor incidence and tumor mass at the site of theimplanted pump (Burkhart et al., 2003, supra). This approach is verylocal however, and a systemic effect of the oligonucleotide onmetastases to distant organ sites remains to be established, in additionto potential toxic side effects on normal cells after systemic delivery.Also, the effect of the oligonucleotide on an already established tumorhas not been shown.

The choice of the target gene is crucial for the development of aneffective neuroblastoma therapy and diagnosis. As mentioned above, thepresent inventors have cloned a novel mRNA splice variant of the DCLKgene, encoding the novel DCL protein, and have functionallycharacterized this splice variant. It was surprisingly found that thissplice variant is exclusively expressed in neuroblastomas, while notbeing detectable in the healthy tissue and cell lines tested. Thisfinding was used to devise novel therapeutic and diagnostic methods.

DEFINITIONS

“Gene silencing” refers herein to a reduction (downregulation) orcomplete abolishment of target protein production in a cell. Genesilencing may be the result of a reduction of transcription and/ortranslation of the target gene. The “target gene(s)” is/are the gene(s)which is/are to be silenced. The target gene is usually an endogenousgene, but may in certain circumstances be a transgene. As methods can beused to silence all or several members of a gene family, the term“target gene” may also refer to a gene family which is to be silenced.

The term “gene” refers to the nucleic acid sequence which is transcribedinto an mRNA molecule (“transcribed region”), operably linked to varioussequence elements necessary for transcription, such as a transcriptionregulatory sequence, enhancers, 5′leader sequence, coding region and3′nontranslated sequence. An endogenous gene is a gene found naturallywithin a cell.

“Sense” refers to the coding strand of a nucleic acid molecule, such asthe coding strand of a duplex DNA molecule or an mRNA transcriptmolecule. “Antisense” refers to the reverse complement strand of thesense strand. An antisense molecule may be an antisense DNA or anantisense RNA, i.e. having an identical nucleic acid sequence as theantisense DNA, with the difference that T (thymine) is replaced by U(uracil).

The term “comprising” is to be interpreted as specifying the presence ofthe stated parts, steps or components, but does not exclude the presenceof one or more additional parts, steps or components. A nucleic acidsequence comprising region X, may thus comprise additional regions, i.e.region X may be embedded in a larger nucleic acid region.

The term “substantially identical”, “substantial identity” or“essentially similar” or “essential similarity” means that two peptideor two nucleotide sequences, when optimally aligned, such as by theprograms GAP or BESTFIT using default parameters, share at least about75%, preferably at least about 80% sequence identity, preferably atleast about 85 or 90% sequence identity, more preferably at least 95%,97%, 98% sequence identity or more (e.g., 99%, sequence identity). GAPuses the Needleman and Wunsch global alignment algorithm to align twosequences over their entire length, maximizing the number of matches andminimizes the number of gaps. Generally, the GAP default parameters areused, with a gap creation penalty=50 (nucleotides)/8 (proteins) and gapextension penalty=3 (nucleotides)/2 (proteins). For nucleotides thedefault scoring matrix used is nwsgapdna and for proteins the defaultscoring matrix is Blosum62 (Henikoff & Henikoff, 1992, Proc. Natl. Acad.Science 89, 915-919). It is clear than when RNA sequences are said to beessentially similar or have a certain degree of sequence identity withDNA sequences, thymine (T) in the DNA sequence is considered equal touracil (U) in the RNA sequence. “Identical” sequences have 100% nucleicacid or amino acid sequence identity when aligned. Also in this case anRNA sequence is 100% identical to a DNA sequence if the only differencebetween the sequences is that the RNA sequence comprises U instead of Tat identical positions. Sequence alignments and scores for percentagesequence identity may be determined using computer programs, such as theGCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685Scranton Road, San Diego, Calif. 92121-3752 USA. Alternatively percentsimilarity or identity may be determined by searching against databasessuch as FASTA, BLAST, etc.

When referring to “sequences” herein or to “sequence fragments”, it isunderstood that molecules with a certain sequence of nucleotides (DNA orRNA) or amino acids are referred to.

“Stringent hybridization conditions” can also be used to identifynucleotide sequences, which are substantially identical to a givennucleotide sequence. Stringent conditions are sequence dependent andwill be different in different circumstances. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequences at a defined ionic strength andpH. The Tm is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Typically stringent conditions will be chosen in which the saltconcentration is about 0.02 molar at pH 7 and the temperature is atleast 60° C. Lowering the salt concentration and/or increasing thetemperature increases stringency. Stringent conditions for RNA-DNAhybridizations (Northern blots using a probe of e.g. 100 nt) are forexample those which include at least one wash in 0.2×SSC at 63° C. for20 min, or equivalent conditions. Stringent conditions for DNA-DNAhybridization (Southern blots using a probe of e.g. 100 nt) are forexample those which include at least one wash (usually 2) in 0.2×SSC ata temperature of at least 50° C., usually about 55° C., for 20 min, orequivalent conditions.

A “subject” refers herein to a mammalian subject, especially to a humanor animal subject.

“Target cell(s)” refers herein to the cells in which DCL protein levelsare to be modified (especially reduced) and include any cancer cells inwhich DCL protein is normally produced, especially neuroblastoma cells.The presence of DCL in target cells can be determined as describedelsewhere herein. Below only neuroblastoma therapy and diagnosis isreferred to, but it is understood that any reference to neuroblastomacells can be applied analogously to other types of cancer target cellsand that such methods, uses and kits are encompassed herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel nucleic acid and protein sequencesfor use in neuroblastoma therapy and diagnostic methods. The DCL proteinwas found to be cell-specifically expressed in all neuroblastoma celllines tested so far (human and mouse cell lines). DCL was found topolymerize and stabilize microtubules and co-localization of endogenousDCL with mitotic spindles in dividing neuroblastoma cells indicates arole of DCL in correct formation of the mitotic spindle in dividingcells. DCL gene silencing in neuroblastoma cell lines resulted indramatic deformation or even absence of the mitotic spindle andmicrotubule disassembly.

The invention thus concerns a method for the treatment of neuroblastoma,said method comprising administering to a subject in need thereof atherapeutically active amount of a nucleic acid fragment of SEQ ID NO: 1or 2, or of a variant of SEQ ID NO: 1 or 2, said nucleic acid fragmentbeing capable of causing a significant reduction of the amount ofDCL-protein of SEQ ID NO: 3 or 4.

Also the invention concerns a composition suitable for the treatment ofneuroblastoma comprising one or more nucleic acid fragments of SEQ IDNO: 1 or 2, or of a variant of SEQ ID NO: 1 or 2, said nucleic acidfragment being capable of causing a significant reduction of the amountof DCL-protein of SEQ ID NO: 3 or 4 when introduced into neuroblastomacells and a pharmaceutically and/or physiologically acceptable carrier.

Nucleic Acid and Amino Acid Sequences According to the Invention

The present invention provides novel nucleic acid sequences, SEQ ID NO:1 (mouse dcl mRNA and cDNA) and SEQ ID NO: 2 (human dcl mRNA and cDNA),which encode the proteins SEQ ID NO: 3 (mouse DCL) and SEQ ID NO: 4(human DCL). The dcl mRNA sequences of SEQ ID NO: 1 and 2 are novelsplice variants of the mouse and human DCLK gene. The splice variantscomprise exon 1 to exon 8 (partially, up to a stop codon), wherein exon1 is non-coding. In both sequences, exon 6 of the DCLK gene is absent.In the mouse mRNA sequence, the translation start codon is found atnucleotides 189-191, while the translation stop codon is found atnucleotides 1275-1277. Exon 2 starts at nt 169, exon 3 starts at nt 565,exon 4 starts at nt 912, exon 5 starts at nt 1012, exon 7 starts at nt1129 and exon 8 starts at nt 1224. In the human mRNA sequence, thetranslation start codon is found at nucleotides 213-215, while thetranslation stop codon is found at nucleotides 1302-1304. Exon 2 startsat nt 194, exon 3 starts at nt 589, exon 4 starts at nt 936, exon 5starts at nt 1036, exon 7 starts at nt 1153 and exon 8 starts at nt1248. The mouse and human DCL proteins are very similar in their aminoacid sequence and both have a molecular weight of about 40 kDa. Themouse DCL protein comprises 362 amino acids, while the human DCL proteincomprises 363 amino acids. Amino acid sequence identity is about 98%, asonly 4 amino acid differences are present. These are at amino acid 172,which is G in the mouse sequence and S in the human sequence, atposition 290 (A in the mouse sequence vs. S in the human sequence), atposition 294 (G in the mouse sequence vs. S in the human sequence) andthe V at position 359 of the human sequence is absent from the mousesequence. Due to the high sequence similarity also at the cDNA/mRNAlevel (which is about 90% for the coding region), either nucleic acidsequence (SEQ ID NO: 1 or 2), or fragments or variants thereof, may beused in gene silencing approaches of target cells, especially of humanneuroblastoma cells.

It is understood that when reference is made herein to an RNA or mRNAmolecule, while the sequence listing depicts a DNA sequence, the RNAmolecule is identical to the DNA sequence with the difference that T(thymine) is replaced by U (uracil).

Apart from the complete nucleic acid sequences of dcl (SEQ ID NO: 1 and2), also sense and/or anti-sense fragments of SEQ ID NO: 1 and 2 areprovided, which are suitable for use in gene silencing methods havingdcl as target gene. The fragment(s) must thus be functional when used inany one of the gene silencing methods described below, and in particularthey cause a significant reduction of the production of the DCL proteinof SEQ ID NO: 3 or 4 when present in neuroblastoma cells. A “significantreduction in the production of SEQ ID NO: 3 or 4” refers to a reductionof the DCL-protein of at least 50%, 60%, 70%, preferably at least 80%,90% or 100% in neuroblastoma cells comprising the sense and/or antisensefragment of SEQ ID NO: 1 and/or 2, compared to the DCL-protein levelfound in neuroblastoma cells into which no sense and/or antisensefragments of SEQ ID NO: 1 and/or 2 were introduced. In addition, theintroduction of the sense and/or antisense fragment of SEQ ID NO: 1and/or 2 causes, by significantly reducing or abolishing DCL-proteinproduction in the cell, a phenotypic change to the cell. In particular,microtubule disassembly and deformation of the mitotic spindle resultsand proliferation of neuroblastoma cells is significantly reduced. A“significant reduction of neuroblastoma cell proliferation” refers to areduction or complete inhibition in growth (cell division) ofneuroblastoma cells comprising the sense and/or antisense fragments ofSEQ ID NO: 1 and/or 2. A skilled person can easily test, using themethods described herein, whether a sense and/or antisense fragment ofSEQ ID NO: 1 and/or 2 has the ability to cause the desired effect. Theeasiest method to test this is to introduce the sense and/or antisensefragments into neuroblastoma cell lines cultured in vitro and analyzedcl mRNA and/or DCL-protein levels and/or phenotypic changes and/orneuroblastoma cell proliferation in those cell, compared to controlcells. The in vitro effect reflects the suitability of the sense and/orantisense fragments to be used to make a composition for the treatmentof neuroblastoma.

In principle, a (sense and/or antisense) fragment of SEQ ID NO: 1 and/or2 may be any part of SEQ ID NO: 1 or 2 comprising at least 10, 12, 14,16, 18, 20, 22, 25, 30, 50, 100, 200, 500, 1000 or more consecutivenucleotides of SEQ ID NO: 1 or 2, or its complement or its reversecomplement. The sense and/or antisense fragment may be an RNA fragmentor a DNA fragment. Further, the fragment may be single stranded ordouble stranded (duplex). The nucleic acid fragment may also be 100%identical to part of the non-coding region of SEQ ID NO: 1 or 2 (e.g. toa region of nucleotides I-188 of SEQ ID NO: 1 or nucleotides 1-212 ofSEQ ID NO: 2), or to part of the coding region (nucleotide 189 to 1274of SEQ ID NO: 1 or nucleotide 213 to 1301 of SEQ ID NO: 2) or to aregion which is partly non-coding and partly coding (such as intron-exonboundaries or exon 1). A nucleic acid fragment may be made de novo bychemical synthesis, using for example an oligonucleotide synthesizer assupplied e.g. by Applied Biosystems Inc. (Fosters, Calif., USA), or maybe cloned using standard molecular biology methods, such as described inSambrook et al. (1989) and Sambrook and Russell (2001). The nucleic acidfragments according to the invention may be used for various purposes,such as: as PCR primers, as probes for nucleic acid hybridization, asDNA or RNA oligonucleotides to be delivered to target cells or as siRNAs(small interfering RNAs) to be delivered to or to be expressed in targetcells. Because different gene silencing methods make use of differentsense and/or antisense nucleic acid fragments, these will, withoutlimiting the scope of the present invention, be described in detailbelow.

In addition, variants of SEQ ID NO: 1 and 2, their complement or reversecomplement, as described above, are provided. “Variants” are not 100%identical in nucleic acid sequence to SEQ ID NO: 1 or 2 (or theircomplement or reverse complement), but are “essentially similar” intheir nucleic acid sequence. “Variants of SEQ ID NO: 1 or 2” includenucleic acid sequences which, due to the degeneracy of the genetic code,also encode the amino acids of SEQ ID NO: 3 or 4, or fragments thereof.Variants of SEQ ID NO: 1 or 2, their complement, reverse complementencompasses also SEQ ID NO: 1 or 2 which differs from SEQ ID NO: 1 or 2through substitutions, deletions and/or replacement of one or morenucleotides. “Variants of SEQ ID NO: 1 and 2” also includes sequencescomprising or consisting of mimics of nucleotides such as PNA's (PeptideNucleic Acid), LNA's (Locked Nucleic Acid) and the like or comprisingmorpholino, 2′-O-methyl RNA or 2′-O-allyl RNA.

Variant nucleic acid sequences may, for example, be made de novo bychemical synthesis, generated by mutagenesis or gene shuffling methodsor isolated from natural sources, using for example PCR technology ornucleic acid hybridization. A variant of SEQ ID NO: 1 or 2 can also bedefined as a nucleic acid sequence which is “essentially similar” (asdefined above) to SEQ ID NO: 1 or 2, their complement or reversecomplement. Especially, variants which have at least 75%, 80%, 85%, 90%,95% or more sequence identity with SEQ ID NO: 1 or 2 over the entirelength of the sequence are encompassed herein. In one embodiment of theinvention sense and/or antisense fragments of nucleic acid sequenceswhich are essentially similar to SEQ ID NO: 1 or 2 are provided. Asdescribed for the fragments of SEQ ID NO: 1 or 2, the fragments ofvariants of SEQ ID NO: 1 or 2 have the ability to significantly reducethe cellular levels of the DCL-protein when introduced in suitableamounts into neuroblastoma cells. Functionally, these variant fragmentsmust therefore be equivalent to the sense and/or antisense fragmentsdescribed, and a skilled person can test the functionality of suchfragments in the same way as described.

Also provided are the isolated proteins of SEQ ID NO: 3 and SEQ ID NO:4, as well as fragments and variants thereof. The DCL proteins (orfragments or variants thereof) according to the invention may forexample be used to raise antibodies, such as monoclonal or polyclonalantibodies, which may then be used in various DCL detection methods,diagnostic or therapeutic methods, or kits. Such antibodies and theiruse in DCL detection methods are an embodiment of the invention.Alternatively, epitopes, which elicit an immune response may beidentified within the proteins. The DCL proteins, fragments or variantsthereof may be made synthetically, may be purified from natural sourcesor may be expressed in recombinant cells or cell cultures. A DCL proteinfragment may be any fragment of SEQ ID NO: 3 or SEQ ID NO: 4 comprising20, 50, 100, 200, or more consecutive amino acids identical oressentially similar to the corresponding part of SEQ ID NO: 3 or 4. DCLprotein variants include amino acid sequences which have substantialsequence identity to SEQ ID NO: 3 or 4, for example amino acid sequenceswhich differ from SEQ ID NO: 3 or 4 by 1, 2, 3, 4, 5 or more amino acidsubstitutions, deletions or insertions. Variants also include proteinscomprising peptide backbone modifications or amino acid mimetics, suchas non-protein amino acids (e.g. β-, γ-, δ-amino acids, β-, γ-, δ-iminoacids) or derivatives of L-α-amino acids. A number of suitable aminoacid mimetics are known to the skilled artisan, they includecyclohexylalanine, 3-cyclohexylpropionic acid, L-adamantyl alanine,adamantylacetic acid and the like. Peptide mimetics suitable forpeptides of the present invention are discussed by Morgan and Gainor,(1989) Ann. Repts. Med. Chem. 24:243-252.

Methods According to the Invention

In one embodiment, the invention provides methods for silencing dclgene(s) in target cells or tissues, especially in neuroblastoma cells.These methods have in common that one or more sense and/or antisensenucleic acid fragments of SEQ ID NO: 1 or 2 or fragments of variants ofSEQ ID NO: 1 or 2 (as described above) is/are delivered to the targetcell(s) (neuroblastoma cells) and is/are introduced into the targetcell(s), whereby the introduction into the target cell(s) results insilencing of the endogenous dcl gene(s) (the target gene), and inparticular results in a significant reduction of DCL-protein andneuroblastoma cell proliferation.

Various gene silencing methods are known in the art. Generally, RNA orDNA with sequence homology to an endogenous target gene is introducedinto a cell with the aim of interfering with transcription and/ortranslation of the endogenous target gene. Production of the targetprotein is thereby significantly reduced or preferably completelyabolished. Known gene silencing methods include antisense RNA expression(see e.g. EP140308B1), co-suppression (sense RNA expression, see e.g.EP0465572B1), delivery or expression of small interfering RNAs (siRNA)into cells (see WO03/070969, Fire et al. 1998, Nature 391, 806-811,WO03/099298, EP1068311, Zamore et al. 2000, Cell 101: 25-33, Elbashir etal. 2001, Genes and Development 15:188-200; Sioud 2004, TrendsPharmacol. Sci. 25:22-28) and antisense oligonucleotide delivery intocells (see e.g. WO03/008543, Pagnan et al. 2000 supra, Burkhard et al.2003, supra). See also Yen and Gerwitz (2000, Stem Cells 18:307-319) fora review of gene silencing approaches.

In addition, various methods for delivering the nucleic acid moleculesto the target cells exist and may be used herein, such as (cationic)liposome delivery (Pagnan et al. 2000, supra), cationic porphyrins,fusogenic peptides (Gait, 2003, Cell. Mol. Life Sci. 60: 844-853) orartificial virosomes (for review see Lysik and Wu-Pong, 2003, J. Pharm.Sci. 92:1559-1573; Seksek and Bolard, 2004, Methods Mol. Biol. 252:545-568).

The cloning and characterization of the mouse and human DCL splicevariant enables the use of any of the known gene silencing methods forsignificantly reducing the DCL protein level (or for completelyabolishing DCL protein production) in mouse or human neuroblastoma cellsin vitro (in cell or tissue culture) or in vivo. Especially, thephenotypic effect of DCL silencing is seen as a deformation of themitotic spindle in dividing neuroblastoma cells and/or a significantreduction or complete inhibition of proliferation of neuroblastoma cellsin vivo or in vitro.

In one embodiment the use of one or more sense and/or antisense nucleicacid fragments of SEQ ID NO: 1 or 2, or fragments of variants of SEQ IDNO: 1 or 2, for the preparation of a composition for the significantreduction of DCL protein levels in neuroblastoma cells, and for thetreatment of neuroblastoma, is provided. In particular, administrationof the composition in suitable amounts and at suitable time intervalsresults in a reduction or complete inhibition of neuroblastoma cellproliferation.

In another embodiment a method for in vitro treatment of neuroblastomacells is provided. This method can be used to test the functionality ofnucleic acid fragments and compositions comprising these. The methodcomprises a) establishment of cell cultures of neuroblastoma cell lines,b) the treatment of the cells with nucleic acid fragments orcompositions comprising the nucleic acid fragments according to theinvention and c) the analysis of phenotypic changes of the neuroblastomacells compared to control cells (cell proliferation, microtubuledisassembly, etc., using visual assessment, microscopy, etc.) and/or themolecular analysis of the cells (analysing dcl transcript levels, DCLprotein levels, etc., using e.g. PCR, hybridization, chemiluminescentdetection methods, etc.).

Non-limiting examples of sense and/or antisense DNA or RNA moleculeswith sequence identity or essential sequence similarity to SEQ ID NO: 1and/or 2, suitable for dcl gene silencing, are the following:

1. Small Interfering RNAs (siRNA)

Small interfering RNAs consist of double stranded RNA (dsRNA) of 18, 19,20, 21, 22, 23, 24, 25, 30, or more contiguous nucleotides of the SEQ IDNO: 1 or 2. Such dsRNA molecules can easily be made synthetically bysynthesizing short single RNA oligonucleotides of the desired sequenceand annealing these subsequently (see Examples). Preferably additionalone, two or three nucleotides are present as 3′overhangs, mostpreferably two thymine nucleotides or thymidine deoxynucleotides (3′-endTT). These dsRNAs comprise both sense and antisense RNA. Non-limitingexamples are the following:

(siDCL-2) 5′-CAAGAAGACGGCUCACUCCTT-3′ (SEQ ID NO: 5)3′-TTGUUCUUCUGCCGAGUGAGG-5′ (SEQ ID NO: 6) (hu-siDCL-2)5′-CAAGAAAACGGCUCAUUCCTT-3′ (SEQ ID NO: 7) 3′-TTGUUCUUUUGCCGAGUAAGG-5′(SEQ ID NO: 8) (siDCL-3) 5′-GAAAGCCAAGAAGGUUCGATT-3′ (SEQ ID NO: 9)3′-TTCUUUCGGUUCUUCCAAGCT-5′ (SEQ ID NO: 10) (hu-siDCL-3)5′-GAAGGCCAAGAAAGUUCGUTT-3′ (SEQ ID NO: 11) 3′-TTCUUCCGGUUCUUUCAAGCA-5′(SEQ ID NO: 12)

As mentioned above, any other fragment of SEQ ID NO: 1 or 2, or of avariant of SEQ ID NO: 1 or 2, may suitably be used to construct siRNAs.siRNA molecules may also comprise labels, such as fluorescent orradioactive labels, for monitoring and detection.

Conveniently, siRNAs may also be expressed from a DNA vector. Such DNAvectors may comprise additional nucleotides between the sense and theantisense fragment, resulting in stem-loop structure, following foldingof the RNA transcript. Instead of delivery and introduction of the siRNAmolecules into neuroblastoma cells such DNA vectors may be transientlyor stably introduced into the target cells, so that the siRNA istranscribed within the target cells. For example, vectors for genedelivery, such as those developed for gene therapy, may be used todeliver DNA into neuroblastoma cells, from which sense and/or antisensefragments of SEQ ID NO: 1 or 2 or of variants of SEQ ID NO: 1 or 2 aretranscribed. Examples are recombinant adeno-associated viral vectors(AAV), as described in Hirata et al., 2000 (J. of Virology74:4612-4620), Pan et al. (J. of Virology 1999, Vol 73, 4: 3410-3417),Ghivizanni et al. (2000, Drug Discov. Today 6:259-267) or WO99/61601.

A skilled person can easily test whether a siRNA molecule is suitablefor, and effective in, dcl gene silencing, by for example delivering themolecule into neuroblastoma cell lines and subsequently assessing dclmRNA and/or DCL protein levels produced by the cells comprising thesiRNA molecule(s), using known methods, such as RT-PCR, NorthernBlotting, nuclease protection assays, Western Blotting, ELISA assays andthe like. Suitable neuroblastoma cell lines are for example human SHSY5,mouse N1E-115, mouse NS20Y or mouse neuroblastoma/rat glioma hybridNG108 lines, or others. Alternatively, phenotypic effects of dcl genesilencing, such as mitotic spindle deformation, can be assessed, asdescribed in the Examples using, for example immunocytochemical stainingor immunofluorescence. Anti-DCL-antibodies can be generated by a skilledperson, e.g. as described in the Examples, or an existing antibody(Kruidering et al. 2001, supra), which was herein found to have a highspecificity for DCL, may be used.

DCL protein levels are preferably reduced by at least about 50%, 60%,70%, 80%, 90% or 100% following introduction of siRNA molecules intoneuroblastoma cells, compared to cells without the siRNA molecules orcompared to cells comprising negative control siRNA molecules, such assiDCL-1 described in the Examples.

2) Antisense RNA Oligonucleotides

Antisense RNA oligonucleotides consist of about 12, 14, 16, 18, 20, 22,25, 30, or more contiguous nucleotides of the reverse complementsequence of SEQ ID NO: 1 or 2. Such RNA oligonucleotides can easily bemade synthetically or transcribed from a DNA vector.

Backbone modifications, such as the use of phosphorothioateoligodeoxynucleotides, may be used to increase the oligonucleotidestability. Other modifications, such as to the 2′sugar moiety, e.g. withO-methyl, fluoro, O-propyl, O-allyl or other groups may also improvestability.

Non-limiting examples of suitable antisense RNA oligonucleotides are:

(DCLex2C) (2′O-methyl RNA phosphorothioate) 5′-GCUGGGCAGGCCAUUCACAC-3′(SEQ ID NO: 13) (hu-DCLex2C) (2′O-methyl RNA phosphorothioate)5′-GCUCGGCAGGCCGUUCACCC-3′ (SEQ ID NO: 14) (DCLex2D) (2′O-methyl RNAphosphorothioate) 5′-CUUCUCGGAGCUGAGUGUCU-3′ (SEQ ID NO: 15)(hu-DCLex2D) (2′O-methyl RNA phosphorothioate)5′-CUUCUCGGAGCUGAGCGUCU-3′ (SEQ ID NO: 16)

As for the siRNA molecules, a skilled person can easily make othersuitable antisense RNA oligonucleotides and test their dcl-genesilencing efficiency as described above. Instead of using contiguousstretches, which match the reverse complement SEQ ID NO: 1 or 2 to 100%,sequences which are essentially similar to the reverse complement of SEQID NO: 1 or 2 may be used, for example by adding, replacing or deleting1, 2 or 3 nucleotides.

Encompassed are also DNA molecules, in particular DNA vectors capable ofproducing antisense RNA oligonucleotides as RNA transcripts or as partof a transcript. Such vectors can be used to produce the antisense RNAoligonucleotides when the vector is present in suitable cell lines. DNAvectors (e.g. AAV vectors, see above) may also be delivered intoneuroblastoma cells in vivo in order to silence endogenous dcl-geneexpression. Thus, instead of delivering antisense RNA oligonucleotides,DNA vectors may be delivered to the neuroblastoma cells and prevent orreduce neuroblastoma cell proliferation.

DCL protein levels are preferably reduced by at least about 50%, 60%,70%, 80%, 90% or 100% following introduction of antisense RNAoligonucleotides into neuroblastoma cells, compared to cells without theantisense RNA oligonucleotides or compared to cells comprising negativecontrol antisense RNA oligonucleotides (i.e. without effect on DCLprotein levels).

3) Antisense DNA Oligonucleotides

Antisense DNA oligonucleotides consist of about 12, 14, 16, 18, 20, 22,25, 30, or more contiguous nucleotides of the reverse complement of SEQID NO: 1 or 2. Such DNA oligonucleotides can easily be madesynthetically.

Backbone modifications, such as the use of phosphorothioateoligodeoxynucleotides, may be used to increase the oligonucleotidestability. Other modifications, such as to the 2′sugar moiety, e.g. withO-methyl, fluoro, O-propyl, O-allyl or other groups may also improvestability.

Non-limiting examples of suitable antisense DNA oligonucleotides are:

(DCLex2A) (DNA phosphorothioate) 5′-GCTGGGCAGGCCATTCACAC-3′ (SEQ ID NO:17) (hu-DCLex2A) (DNA phosphorothioate) 5′-GCTCGGCAGGCCGTTCACCC-3′ (SEQID NO: 18) (DCLex2B) (DNA phosphorothioate) 5′-CTTCTCGGAGCTGAGTGTCT-3′(SEQ ID NO: 19) (hu-DCLex2B) (DNA phosphorothioate)5′-CTTCTCGGAGCTGAGCGTCT-3′ (SEQ ID NO: 20)

As for the siRNA molecules and antisense RNA oligonucleotides, a skilledperson can easily make other suitable antisense DNA oligonucleotides andtest their dcl-gene silencing efficiency as described above.

Instead of using contiguous stretches, which match the reversecomplement of SEQ ID NO: 1 or 2 to 100%, sequences which are essentiallysimilar to the reverse complement of SEQ ID NO: 1 or 2 may be used, forexample by adding, replacing or deleting 1, 2 or 3, or more nucleotides.

DCL protein levels are preferably reduced by at least about 50%, 60%,70%, 80%, 90% or 100% following introduction of antisense DNAoligonucleotides into neuroblastoma cells, compared to cells without theantisense DNA oligonucleotides or compared to cells comprising negativecontrol antisense DNA oligonucleotides (i.e. without effect on DCLprotein levels).

It is understood, that delivery of mixtures of siRNA molecules,antisense RNA oligonucleotides and/or antisense DNA oligonucleotides mayalso be used for dcl specific silencing.

The compositions according to the invention thus comprise a suitableamount of a sense and/or antisense fragment of SEQ ID NO: 1 or 2 or of asequence essentially similar to SEQ ID NO: 1 or 2 and a physiologicallyacceptable carrier. When the compositions are used for introduction intoneuroblastoma cell cultures in vitro, the composition may also comprisea targeting compound, although the presence of a targeting compound isnot required, as the molecules may be introduced simply by transfectionusing for example transfection kits available (e.g. Superfect, Qiagen,Velancia, Calif.), electroporation, liposome mediated transfection, andthe like. A “targeting compound” refers to a compound or molecule whichis able to transport the nucleic acid fragments in vivo to the targetneuroblastoma cells, i.e. it has cell-targeting capabilities.

A “suitable amount” or a “therapeutically effective amount” refers to anamount which, when present in a neuroblastoma cell, is able to cause DCLprotein levels to be significantly reduced or abolished and to causeneuroblastoma cell proliferation to be significantly reduced orinhibited completely. A suitable amount can be easily determined by askilled person without undue experimentation, as described. Suitableamounts of the sense and/or antisense molecules (siRNA, antisense RNA orDNA oligonucleotides) range for example from 0,05 μmol to 5 μmol per mland is infused at 1 to 100 ml per kg body weight.

Compositions which are to be administered to a subject, rather than toneuroblastoma cell cultures, comprise a therapeutically effective amountof the nucleic acid molecules of the invention and in addition one ormore targeting compounds. Such targeting compounds may, for example, beimmunoliposomes, such as described by Pagnan et al. (2000, supra) or byPatorino et al. (Clin Cancer Res. 2003, 9(12):4595-605). Immunoliposomescomprise cell surface-directed antibodies on their exterior. Forexample, monoclonal antibodies raised against antigens of neuroblastomacells, such as the disialoganglioside GD₂ antigen, may be used to targetthe liposomes to neuroblastoma cells. Clearly, other neuroblastoma cellantigens may be used to raise cell specific antibodies. The nucleic acidmolecules are encapsulated in the immunoliposomes using known methodsand the monoclonal antibodies are covalently coupled to the exterior ofthe liposomes (see e.g. p254 of Pagnan et al. 2000, supra). The bindingof the liposomes to neuroblastoma cells and the uptake of the nucleicacid molecules by the neuroblastoma cells can be assessed in vitro usingknown methods, as described in Pagnan et al. (2000). Similarly,phenotypic effects and/or molecular effects of the intracellularpresence of the nucleic acids can be assessed.

Other targeting compounds may be antibodies as such, for examplemonoclonal antibodies raised against a neuroblastoma cell surfaceantigen conjugated to the nucleic acid molecules. For example ananti-transferrin-receptor antibody may be used, such as the chimericrat/mouse monoclonal antibody ch17217 which has been shown to targetcytokines to neuroblastoma tumor cells in mice (Dreier et al., 1998,Bioconj. Chem. 9: 482-489). Such methods are well known in the art, seee.g. Guillemard and Saragovi (Oncogene, Advanced online publication,published 22 Mar. 2004, Prodrug chemotherapeutics bypass p-glycoproteinresistance and kill tumors in vivo with high efficacy andtarget-dependent selectivity).

Similarly, the nucleic acid molecules according to the invention may beconjugated to natural or synthetic ligands, or ligand mimetics, whichbind to the target cell surface receptors (e.g. neuroblastoma cellsurface receptors) and which result in the endocytosis of the nucleicacid molecules. An example of such a ligand is for example transferrin.It has been shown that intravenous injection of transferrin-PEG-PEI/DNAcomplexes resulted in gene transfer to subcutaneous Neuro2aneuroblastoma tumors in mice (Ogris et al., 2003, J. Controlled Release91: 173-181).

The therapeutic composition may further comprise various othercomponents, such as but not limited to water, saline, glycerol orethanol. Additional pharmaceutically acceptable auxiliary substances maybe present, such as emulsifiers, wetting agents, buffers, tonicityadjusting agents, stabilizers and the like, for example, sodium acetate,sodium lactate, sodium chloride, potassium chloride, calcium chloride,sorbitan monolaurate, and triethanolamine oleate. Other biologicallyeffective molecules may be present, such as nucleotide molecules whichsilence other gene targets (e.g. c-Myb), markers or marker genes (e.g.luciferase), ligands, antibodies, drugs, etc.

The therapeutic compositions may be administered locally, e.g. byinjection, preferably into the target tissue, or systemically, e.g. bydropwise infusion of a parenteral fluid or a subcutaneous slow releasedevice.

Injectable delivery systems include solutions, suspensions, gels,microspheres and polymeric injectables, and can comprise excipients suchas solubility-altering agents (e.g. ethanol, propylene glycol andsucrose) and polymers (e.g. polycaprylactones, and PLGA's). Furtherguidance regarding formulations that are suitable for various types ofadministration can be found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985).

In one embodiment the compositions according to the invention are usedto complement other neuroblastoma therapies, such as chemotherapy,radiation therapy, surgery and/or bone marrow transplantation. Thus,either before, at the same time and/or shortly after one or moreconventional treatments, the compositions are administered to thesubject, preferably weekly, more preferably monthly, in effectiveamounts. Any neuroblastoma cells, which are not effectively removed oreradicated by the other therapy are thus prevented from proliferating bydcl silencing. This treatment reduces the risk of spread ofneuroblastoma cells to other parts of the body (metastasis formation)and prevents or at least delays relapses, i.e. the recurrence of the(primary) neuroblastoma. DCL silencing has as advantage overchemotherapy or surgery that it has a low toxicity towards normal tissueand a high specificity for neuroblastoma cells. It is therefore likelythat undesirable side effects are absent or minimal.

In another embodiment a method for treatment of a subject is provided,whereby no other neuroblastoma therapies (e.g. chemotherapy, surgery,etc.) are carried out. The method comprises a) establishing a diagnosisof neuroblastoma, and b) administering a suitable amount of acomposition according to the invention, and c) monitoring at variousintervals (follow up treatment).

Step a), diagnosis, can, for example, be established using thediagnostic method and kits described below. Alternatively, neuroblastomadiagnosis may be established using conventional methods, such as CT orCAT scans, MRI scans, mIBG scan (meta-iodobenzylguanidine), X-rays,biopsies or analysis of catecholamines or its metabolites in urine orblood plasma samples (e.g. dopamine, homovanillic acid, rvanillylmandelic acid). Step b) is described elsewhere herein. Step c)may involve various follow up tests, such as the diagnostic testdescribed below, blood or urine tests, CT scans, MRI scan, etc. Thepurpose of the follow up monitoring is to ensure that the tumor cellsare completely eradicated and do not recur. If this is not the case, newtreatment needs to be started.

In a further embodiment diagnostic methods and diagnostic kits areprovided which are useful for selective screening of early stageneuroblastoma occurrence in subjects. Subjects may already have testedpositive in one or more other neuroblastoma tests, in which case thepresent test may confirm earlier diagnosis. Alternatively, they may nothave been diagnosed with neuroblastoma yet, but they may show symptomswhich could be caused by neuroblastoma. Depending on the tumor location,symptoms may vary greatly, such as loss of appetite, tiredness,breathing or swallowing difficulties, swollen abdomen, constipation,weakness/unsteadiness in the legs, etc. Alternatively, high risksubjects not showing any symptoms yet may be prophylactically tested atregular intervals using the diagnostic method according to the inventionto ensure early diagnosis, which greatly increases the chances oferadication of the neuroblastoma cells. The ex vivo diagnostic methodscomprise taking a blood sample from a subject and detecting the presenceor absence of free neuroblastoma cells in the serum. Alternatively, theex vivo diagnostic method may be carried out on a biopsy sample of the(presumed) tumor tissue. As the DCL protein and the dcl mRNA arespecific for neuroblastoma cells, the presence of the cells can bedetected, and optionally quantified, by analysing the presence of dclmRNA and/or DCL protein in the sample. This can be done using methodsknown in the art, such as (quantitative) RT-PCR using dcl-specific ordegenerate primers, other PCR methods, such as for example specificamplification of regions of the dcl gene, DNA-arrays, DNA probes forhybridization, or methods which detect the DCL protein, such asEnzyme-linked immunosorbent assays (ELISA) or Western blotting usingDCL-specific antibodies (e.g. monoclonal or polyclonal antibodies). Inone embodiment the diagnostic method and kit according to the inventioncomprises the monoclonal antibody anti-DCLK (also referred to asanti-CaMLK in Kruidering et al. 2001, supra), which recognizes and bindshuman and/or mouse DCL protein (detectable as having a molecular weightof about 40 kDa) according to the invention. Although anti-DCLK alsorecognizes other splice variants, such as DCLK-short (i.e. cpg16) andCARP, the spatio-temporal separation of DCL from cpg16 and CARPexpression, and the differences in molecular weight, can be used toeasily minimize/avoid false positives. Clearly, other DCL-specificmonoclonal antibodies may be generated and used.

Also, primers or probes specific for exon 8 RNA (present in DCL RNA butabsent in DCLK-short RNA) may be used in RNA detection methods. Ascontrols, for example primers or probes which bind to (hybridize with)exon 6 RNA of DCLK-short (i.e. cpg16) and CARP, or to exon 9 to 20 ofDCLK may be employed, which are absent in DCL RNA.

Primer pairs, probes and antibodies which specifically detect (e.g. bysequence specific amplification, by sequence specific hybridization orby specific binding) the RNA or DNA of SEQ ID NO: 2 or the protein ofSEQ ID NO: 4 can be made by a skilled person using standard molecularbiology methods, as found in references to standard textbooks below.Primer pairs and probes can be made on the basis of SEQ ID NO: 2.Monoclonal or polyclonal antibodies specific for DCL-protein can beraised as known in the art.

The diagnostic method comprises the steps of a) analyzing a blood sampleof a subject for the presence or absence of SEQ ID NO: 2 RNA or DNAand/or for the presence or absence of DCL protein of SEQ ID NO: 4 and b)optionally quantifying the amount of SEQ ID NO: 2 and/or SEQ ID NO: 4present. A quantification may allow a direct correlation to the numberof neuroblastoma cells present, which in turn may indicate the severityof the neuroblastoma development and spread.

Also provided are ex vivo diagnostic kits for carrying out the methodabove. A diagnostic kit may, therefore, comprise primers, probes and/orantibodies, and other reagents (buffers, labels, etc.), suitable for dclgene, dcl mRNA and/or DCL protein detection and optionallyquantification. In addition, kits comprise instructions and protocolshow to use the reagents (e.g. immunodetection reagents) and controlsamples, for example isolated DCL-protein or dcl DNA.

The following non-limiting examples illustrate the identification,isolation and characterization of the novel DCL splice variant. Unlessstated otherwise, the practice of the invention will employ standardconventional methods of molecular biology, virology, microbiology orbiochemistry. Such techniques are described in Sambrook and Russell(2001) Molecular Cloning: A Laboratory Manual, Third Edition, ColdSpring Harbor Laboratory Press, NY, in Volumes 1 and 2 of Ausubel et al.(1994) Current Protocols in Molecular Biology, Current Protocols, USAand in Volumes I and II of Brown (1998) Molecular Biology LabFax, SecondEdition, Academic Press (UK), Oligonucleotide Synthesis (N. Gaiteditor), Nucleic Acid Hybridization (Hames and Higgins, eds.), “Enzymeimmunohistochemistry” in Practice and Theory of Enzyme Immunoassays, P.Tijssen (Elsevier 1985). Standard materials and methods for PCR can befound in Dieffenbach and Dveksler (1995) PCR Primer: A LaboratoryManual, Cold Spring Harbor Labroatory Press, and in McPherson et al(2000) PCR Basics: From Background to Bench, first edition, SpringerVerlag Germany. Methods for making monoclonal or polyclonal antibodiesare for example described in Harlow and Lane, Using Antibodies: Alaboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1998,and in Leddell and Cryer “A Practical Guide to Monoclonal Antibodies”,Wiley and Sons 1991. All above references are incorporated herein byreference.

Throughout the description and Examples reference to the followingsequences is made:

-   SEQ ID NO 1: cDNA sequence of mouse dcl-   SEQ ID NO 2: cDNA sequence of human dcl-   SEQ ID NO 3: amino acid sequence of mouse DCL-   SEQ ID NO 4: amino acid sequence of human DCL-   SEQ ID NO 5: siDCL-2 sense RNA oligonucleotide (siRNA strand)-   SEQ ID NO 6: siDCL-2 antisense RNA oligonucleotide (siRNA strand)-   SEQ ID NO 7: hu-siDCL-2 sense RNA oligonucleotide (siRNA strand)-   SEQ ID NO 8: hu-siDCL-2 antisense RNA oligonucleotide (siRNA strand)-   SEQ ID NO 9: siDCL-3 sense RNA oligonucleotide (siRNA strand)-   SEQ ID NO 10: siDCL-3 antisense RNA oligonucleotide (siRNA strand)-   SEQ ID NO 11: hu-siDCL-3 sense RNA oligonucleotide (siRNA strand)-   SEQ ID NO 12: hu-siDCL-3 antisense RNA oligonucleotide (siRNA    strand)-   SEQ ID NO 13: DCLex2C antisense RNA oligonucleotide-   SEQ ID NO 14: hu-DCLex2C antisense RNA oligonucleotide-   SEQ ID NO 15: DCLex2D antisense RNA oligonucleotide-   SEQ ID NO 16: hu-DCLex2D antisense RNA oligonucleotide-   SEQ ID NO 17: DCLex2A antisense DNA oligonuclotide-   SEQ ID NO 18: hu-DCLex2A antisense DNA oligonuclotide-   SEQ ID NO 19: DCLex2B antisense DNA oligonuclotide-   SEQ ID NO 20: hu-DCLex2B antisense DNA oligonuclotide

FIGURE LEGENDS

FIG. 1.—Genomic Organization of DCL and Alignment with DCX

(A): Genomic organization of the DCLK gene and the cloning strategy ofthe DCL cDNA. Only the exon-intron structure of the DCL part isindicated including the recently identified exon 8 encoding the common3′ end of CARP and DCL (Vreugdenhil et al., 2001, supra). Exons arerepresented by rectangles and indicated by arabic numbers; introns aresolid lines. The DCL transcript is indicated below (DCL) the genomicstructure. The ORF is represented by a rectangle, non-translatedsequences by lines. The location of the primers, used to clone DCL, isindicated by arrows.

(B): Alignment of the DCL (SEQ ID NO: 3) protein with DCX (SEQ ID NO:28). Identical residues are dark grey and conserved substitutions arelight grey. The two DCX domains and the SP-rich domain

FIG. 2.—DCL is a M.A.P. and Stabilizes Microtubules

Panel I:

(A-C) DCL overexpression in COS-1 cells.

(D-F) DCL overexpressed in COS-1 cells treated with colchicine.

Green represents DCL; red represents α-tubulin and yellow indicates DCLcolocalization with α-tubulin. Blue represent DNA (nucleus). Arrowsindicate DCL associated microtubule bundles, which are resistant tocoichicine treatment. Also note the clear association with a centrosomein A and B. Scale bar is 10 μm.

Panel II. Microtubules polymerization in vitro by DCL. Differentconcentrations of recombinant DCL protein were incubated with purifiedtubulin and the turbidity of the DCL/tubulin mixture was monitored at340 nm for 30 min. Taxol was used as a positive control and water as anegative control. The graph shown is a typical example from multipleexperiments (N=4) with similar results.

FIG. 3.—Expression of DCL is Developmentally Regulated

(A): Cross-reactivity of DCX and DCLK recognizing antibodies. Westernblot analysis of lysates of COS-1 cells overexpressing DCL (lane 4-6) ortwo different DCX variants (lane 2 and 3) with anti-DCX (upper panel) oranti-DCLK (lower panel). Anti-DCLK strongly recognizes DCL (lane 4-6).

(B): Onset of DCL and DCX protein during embryogenesis. Immunoblots ofembryonic brain fractions from age ED8 to ED18 and adult were stainedwith anti-DCLK and anti-DCX. As a positive control for the CARP/DCLantibody, an extract of COS-1 cells overexpressing DCL was used.

(C): Western blot analysis of DCL and DCX in various adult brainregions. 1: ED12 head (positive control), M: Molecular weight marker 2:cerebellum, 3: brain stem, 4: hypothalamus, 5: cerebral cortex, 6:hippocampus 7: olfactory bulb.

FIG. 4.—Localization and Ontogeny of DCL Expression in Embryonic Brain

I.: In situ hybridization of a transversal brain section of embryonicday (ED) 8 (panel A); and sagittal sections at ED10 and ED12 (panel Band C, respectively). On ED8 and ED10, the signal was low but increasedconsiderably at ED12. Abbreviations: di—diencephalon, lv—lateralventricle, me—mesencephalon; mo—medulla oblongata, mt—metencephalon,mv—mesenchephalic vesicle, nc—neopallial cortex, ne—neuroepithelium,rh—rhombencephalon, te—telencephalon, tv—telencephalic vesicle, IV v—4thventricle. Scale bar: 1 mm; exposure time: 14 days).

II: DCL protein distribution in the early mouse neuroepithelium.

A: DCL protein distribution at ED11 (sagittal section). Staining isrestricted to the proliferative regions (telencephalon and diencephalonon the left and right, respectively) and found in the outer layers closeto the pia as well as in the inner ventricular zone (arrowheads; seealso higher magnifications below), whereas normeuronal tissue like themandibular component of the first branchial arch (M) is devoid of anysignal. IV; fourth ventricle. Bar represents 150 μm.

B+C: Adjacent transversal (coronal) sections from the earlyneuroepithelium at ED 9 immunostained for DCX (B) and DCL (C). No DCXstaining is observed (arrowheads in B), whereas DCL is already expressedboth in the inner ependymal (upper 2 arrows) as well as in outer,marginal region (lower arrow). Bar represents 25 μm.

D: Sagittal section of the neuroepithelium of the neural tube at ED11,showing abundant expression at the luminal border (arrowheads), while inthe developing neuronal tissue, isolated dividing cells areimmunopositive as well (arrows). L indicates the neural lumen of theneural tube. Bar represents 70 μm.

E: Detail of a DCL-immunopositive mitotic cell in the neuroepithelium.The chromosomes (arrowhead) oriented in the midline cleavage plane areobvious. Bar represents 3 μm.

F: Overview of the neuroepithelium of the telencephalon at ED10, showingDCL expression in the ventricular (ependymal) layer (arrowhead on theleft) as well as on the marginal/cortical plate zone (arrowhead on theright). 2 immunopositive doublets of dividing cells in the intermediatezone are also visible (arrows). Bar represents 15 μm.

G+H: Transversal cross-sections of the cortical neuroepithelium,illustrating the differential, yet partly overlapping, distributions ofDCX and DCL. DCX is not expressed until ED11 (G), and mainly in theuppermost part of the cortical plate and marginal/cortical plate region(arrow) of the cortical neuroepithelium. DCL, in contrast, is alreadyexpressed at ED9 (H) at particularly high levels in the ventricular(ependymal) layer (arrowhead to the left) with lower levels in theintermediate and marginal zones (arrowhead to the right). Note that theventricular layer (asteriks in G) is devoid of DCX signal. Barrepresents 5 μm.

I: Detail of the ependymal layer of the ventricular zone at ED9 showingDCL Expression in fibers extending from the neuroepithelium into theintermediate zone (arrowheads). Bar represents 12 μm.

J: Detail of the ependymal layer showing clear immunoreactivity individing neuroepithelial cells adjacent to the lumen, that are in;telophase (left), anaphase (middle), while also a DCL positive cell inmid prophase is visible that appears to divide vertically (arrowheads)while migrating away from the lumen (right). Bar represents 8 μm.

K: DCL immunoreactivity in the ependymal layer at ED11, in cells inprophase and telophase (arrowheads) as well as in a blast-like cell inmetaphase/anaphase (arrow). Bar represents 10 μm.

L: Two DCL immunopositive mitotic cells in the ependymal layerdisplaying intense immunoreactivity also in the centrosome-likestructures (lower arrows). Bar represents 1.5 μm.

M+N: Examples of 2 DCL immunopositive, dividing cells in anaphaseII/telophase II (M) and in metaphase/anaphase I, with the chromosomesclearly visible (arrow), while also some microtubular staining isobserved (arrowheads). Bars represent 1 μm.

FIG. 5.—DCL expression in Neuroblastoma cells.

Panel I:

A: DCL is endogenously expressed in several neuroblastoma cell-lines.Screening by Western Blot analysis for DCL positive cell lines. Lane 1:COS-1 cells, lane 2: Hela cells, lane 3: NG108-15 cells, lane 4: NS20Ycells, lane 5: N1E-115 cells, lane 6: molecular weight marker, lane 7:SHSY5 cells. Note that DCL is expressed in neuroblastoma cell lines(lane 3, 4, 5 and 7) but not in cell lines from non-neuronal origin(lane 1 and 2).

B: DCL is a phosphoprotein. NG108-15 lysates stained with anti-DCL. Lane1: untreated lysate, lane 2: lysate incubated at 37° C. withoutphosphatase, lane 3: lysate incubated at 37° C. with phosphatase. Lane4-6 are similar as 1-3 but with DCL overexpression. Note that endogenousDCL comigrates with overexpressed DCL in lane 4-6.

Panel II:

Western blot analysis of DCL expression in N1E-115 cells with (1 to 3)and without (4) siRNA treatment performed in duplo. Three differentsiRNA molecules targeting DCL were used: siDCL-1 (lanes 1), siDCL-2(lanes 2) and siDCL-3 (lanes 3). Note that siDCL-2 and 3 lead to aneffective knock-down while siDCL-1 failed to do so. As a reference, thesame membrane was re-stained with α-tubulin.

Panel III.

Knock-down of DCL leads to relaxation of the microtubule cytoskeleton ininterphase. Anti-DCLK (green) staining yields a spickled pattern, whichis most prominent near the nucleus (A) in non-treated cells (A-C). Thispattern is not affected by siDCL-1 (D) but anti-DCLK staining is almostabsent by effective DCL knockdown by siDCL-3 (G). The cytoskeleton, asindicated by α-tubulin staining (B, E and H), has a fine-maze structurein non-treated cells (B) and in cells treated with si-DCL-1 (E) but isgreatly relaxed by siDCL-3. Merged illustrations of DCL and α-tubulinstaining show non-treated cells (C), cells transfected with siDCL-1 (F)and cells transfected with siDCL-3 (I). Green=DCL, Red=α-tubulin,Yellow=colocalization of DCL and α-tubulin. Scale bar is 10 μm.

Panel IV:

DCL knock-down does not affect centrosome structure. Spickled anti-DCLKstaining (A, D, G) is highly concentrated (A, D) around centrosomes asindicated by anti-γ-tubulin staining (B, E and H) and effectiveknock-down of DCL (G) does not lead to obvious changes in the structureor form of centrosomes (I). Merged illustrations of DCL and α-tubulinstaining are shown of non-treated cells (C), cells transfected withsiDCL-1 (F) and cells transfected with siDCL-3 (I). Green=DCL,Red=γ-tubulin, Yellow=colocalization of DCL and γ-tubulin. Scale bar is10 μm.

FIG. 6.—DCL knock-down leads to deformation of mitotic spindles. Innon-treated cells (A-C) DCL (A) largely colocalizes with α-tubulin (B).The merged image (C) indicates DCL presence at the kinetochore (arrow).Transfection with siDCL-1 (D-F) did not lead to a DCL knockdown (D) andalso did not change the formation of mitotic spindles as indicated byα-tubulin staining (E). Effective DCL knockdown by siDCL-2 (G-I) orsiDCL-3 (J-L) lead to a disappearance of DCL (G, J) and to thedisappearance (H) and deformation (K) of mitotic spindles as indicatedby α-tubulin staining. Green=DCL, Red=α-tubulin, Yellow=colocalizationof DCL and α-tubulin. Scale bar is 10 μm.

FIG. 7-DCL overexpression in dividing COS-1 cells.

A-C: Immunocytochemical analysis of DCL overexpression. A normaldividing COS-1 cell stained with α-tubulin is shown as reference (ref).Overexpression of DCL (Green, A) leads to elongation of mitotic spindlesas indicated by co-staining with α-tubulin (B). Note the difference inmitotic spindle length, indicated by arrows, of transfected versusnontransfected cells. DNA is stained with DAPI (blue).

D-I: Confocal microscopy of DCL overexpression in COS-1 cells duringcell division. One phenotype looks similar (D-F) to wildtype COS-1 cellsin which DCL (D) largely colocalizes with α-tubulin (E). Similar toendogenous localization of DCL in dividing N1E-115 cells, DCL also islocated at the kinetochore. DCL localization is shown in green, whichoverlaps with mitotic spindles as indicated by α-tubulin staining (red).The other phenotype observed lead to elongation and altered orientationof the mitotic spindles (G-I). Green=DCL (A, D, G), Red=α-tubulin(B,E,H), Yellow=colocalization of DCL and α-tubulin (C, F, I). Scale baris 10 μm.

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee

EXAMPLES Example 1 Cloning of DCL from Mouse and Human

DNA sequence analysis of a DCL cDNA clone from mouse (SEQ ID NO: 1)revealed an open reading frame of 362 amino acids (SEQ ID NO: 3) with apredicted molecular mass of 40 kDa (FIG. 1B) and 73% amino acid identity(81% similarity) with mouse DCX over the entire length of both proteins.Alignment of the two predicted DCX repeats (Taylor et al., 2000, supra)with mouse DCX revealed an even higher amino acid identity of 81% (89%similarity) for DCX domain 1 and 90% amino acid identity (99%similarity) for DCX domain II, strongly suggesting that this latterdomain has a similar function in both proteins. The serine/proline(SP)-rich C-terminus, which corresponds largely with CARP (Vreugdenhilet al., 1999, Neurobiology 39, 41-50), exhibits a lower amino acididentity of 63% (78% similarity). This SP-rich domain is present in bothDCX and DCL. Such SP-rich domains are potential MAP kinase motifs(Sturgill et al., 1988, Nature 334, 715-718), suggesting that theC-terminus is a MAP kinase substrate. Interestingly, the YLPL motif inthis region of DCX has been shown to interact with AP-1 and AP-2 and hasbeen implicated in protein sorting and vesicle trafficking (Friocourt etal., 2001, Mol. Cell Neurosc. 18, 307-319). In DCL, however, thecorresponding motif is YRPL in which a hydrophobic leucine is replacedby a basic arginine residue, indicating that DCL is not likely tointeract with AP-1 and AP-2.

The human dcl cDNA/mRNA (SEQ ID NO: 2) and protein (SEQ ID NO: 4)sequences were obtained from a human neuroblastoma cell line (SHSY5)using the mouse sequences and were found to be very similar to themurine sequences, as described elsewhere herein.

Example 2 DCL is a MAP (Microtubule Associated Protein) and Stabilizesthe Cytoskeleton

The two DCX domains of both DCX and DCLK-long have been shown tointeract with and to stabilize microtubule structures (Francis et al.,1999, supra; Gleeson et al., 1999 supra; Kim et al., 2003, Struct. Biol.10, 324-333; Lin et al., 2000, supra). As DCL contains DCX domains thatare identical to DCLK-long, a similar stabilizing and polymerizingeffect on microtubules was expected for DCL. To confirm this, threetypes of experiments were conducted: first, overexpression of DCL inCOS-1 cells resulted in a fibrillar staining pattern in the somaoverlapping the microtubule distribution (FIG. 2.I A), as shown byco-localization with α-tubulin antibodies (FIGS. 2.I B and C). Second,to test if DCL-containing microtubule bundles exhibit a similarresistance to depolymerization as is known for DCX and other MAPs, DCLtransfected cells were exposed to 10 μg colchicine, a compound whichdepolymerizes and disrupts tubulin microtubules. Non-transfected cellsexhibited clear depolymerization of the microtubule cytoskeleton,whereas the microtubule cytoskeleton of all DCL transfected cells wasresistant to 1 hr colchicine treatment, in particular in condensedmicrotubule/DCL bundles (FIG. 2.I D-F). This showed that DCL, similar toDCLK-long and DCX, is capable of stabilizing microtubules. Third, in anin vitro polymerization assay the microtubule polymerizing properties ofDCL were tested by incubating different concentrations of recombinant,non-tagged DCL with purified tubulin. Taxol was used as a positivecontrol, which is a well-known microtubule polymerizing compound.Spectrophotometrical monitoring of microtubule polymerization revealedthat DCL polymerizes microtubules in a dose-dependent manner (FIG.2.II). Together, these data showed that DCL, like DCLK-long and DCX, candirectly polymerize and stabilize microtubules.

Example 3 Characterization of a DCL Recognizing Antibody

Recently the generation of an antibody against CARP, called anti-CaMKLK,has been described (Kruidering et al., 2001, supra) which alsorecognizes other splice-variants of the DCLK gene including DCLK-short(also known as cpg16 (Silverman et al., 1999, J. Biol. Chem. 274,2631-2636) or CaMLK). CARP is a small protein of 55 amino acids of which43 are identical with the C-terminus of DCL, that shares 70% amino acidhomology with human DCX (Vreugdenhil et al., 1999). To address thespecificity of anti-CaMLK, DCX and DCL were overexpressed in COS-1 cellsand analysed for possible cross-reactivity by Western Blot analysis.Anti-CaMLK strongly recognized DCL (FIG. 3A lane 4-6) whereas only somecross-reactivity was observed with DCX (FIG. 3A lane 2 and 3). On theother hand, the DCX antibody used herein, raised against the C-terminal17 amino acid of DCX, strongly recognized DCX (FIG. 3A lane 2 and 3) andnot DCL (FIG. 3A lane 4-6). Thus, anti-CaMLK strongly recognizesnumerous splice variants of the DCLK gene including DCLK-short and DCLand therefore is herein referred to as “anti-DCLK”. In addition, somecross-reactivity of anti-DCLK with DCX may occur, whereas the DCXantibody is specific for DCX alone and not for DCL.

Example 4 DCL is Highly Expressed at Early Stages of Brain Development

Western blot analysis of embryonic brain homogenates revealed thepresence of a 40 kDa protein immunopositive for anti-DCLK. The size ofthis protein corresponds with that of the recombinant DCL proteinoverexpressed in COS-1 cells (FIG. 3B). Anti-DCLK recognizes only DCL inthe developing mouse brain as no other immuno-reactive bands wereobserved (FIG. 3B lane 8-18). Although signal was already present atED10, highest levels of immunoreactive DCL protein were found at ED12and ED14. The level of DCL protein declined after ED14 and a weaker butclear 40 kDa band was still present in adult brain. Here, an additionalband of 53 kDa was very prominent (FIG. 3B). In agreement with itsmolecular weight of 53 kDa, this band most likely representedDCLK-short, which is abundantly expressed only in adult and notdeveloping brain (Vreugdenhil et al, 2001; Omori et al., 1998). Withinthe adult brain, highest levels of DCL protein were found in theolfactory bulb, with lower levels in the hippocampus and cerebralcortex, and very low levels in cerebellum, brain stem and hypothalamus(FIG. 3C).

DCX has been reported to be specifically expressed during developmentbut to drop below detection level in adult brain (Francis et al., 1999supra; Gleeson et al., 1999 supra), although DCX remains expressed invery low amounts in selected regions (Nacher et al., 2001, Eur JNeurosci 14, 629-644). For comparison to the DCL findings, the proteinlysates were further analysed with a DCX-specific antibody recognizingthe C-terminus. In agreement with other studies (Francis et al., 1999;Gleeson et al., 1999), the highest concentrations of DCX were found atED12 and were found to decline afterwards (FIG. 3B).

In contrast to DCL, no DCX protein expression, also after prolongedexposure, could be detected in embryo heads of ED8 and ED10 or in theadult brain. However, consistent with a role for DCX in neuronalmigration, DCX immunoreactivity was observed in the adult olfactory bulb(FIG. 3C lane 7), but not in other brain structures (FIG. 3C lane 2-6),indicating dilution of DCX below detection level in the whole brainlysates.

To analyze regional differences in DCX and DCL expression in moredetail, the spatio-temporal expression of DCL during early embryonicdevelopment was studied using in situ hybridization. Low levels of DCLmRNA expression were observed along the length of the neuroepithelium(destined to give rise to the central nervous system and the layeredcortex in later stages of development) at ED8 (FIG. 4.I A). At ED10,when massive divisions start to become prominent, substantial expressionwas found in the early diencephalon, telencephalon and mesencephalon,a.o. (FIG. 4.I B). Consistent with the RT-PCR and Western blotexperiments the intensity of DCL expression at ED12 increased profoundly(FIG. 4.I C) as compared to ED 8 and 10, with high levels in theproliferative ventricular zones.

To study the spatio-temporal distribution of DCL protein,immunohistochemistry was performed on sections from mouse embryos at 8,9, 10, and 11 days old using the DCLK antibody (anti-DCLK) thatrecognizes DCL exclusively at these ages (see above and FIG. 3B). AtED8, no DCL staining was observed (data not shown). However, at ED 9, anage at which no DCX protein expression is found yet (FIG. 4.II B), DCLsignal was prominent in the ventricular walls and main neuroepithelia,well-defined areas of massive mitosis and neurogenesis (FIG. 4.II C andJ). At ED10 and 11, DCL protein generally followed the in situhybridization pattern, with high levels in the proliferative regions ofthe central and peripheral nervous system, including the telencephalon,diencephalon, lateral ganglionic eminence, the neuroepithelium of theneural tube, as well as e.g. the dorsal root and sympathetic ganglia,whereas non-neuronal tissues like bone or the intestines e.g., weredevoid of any signal (FIG. 4.II A and D). Higher magnifications of theearly neocortex, revealed DCL expression not only in the upper layers ofthe cortical plate, but also in the inner ventricular zone, with lowerlevels apparent in the intermediate zone (FIG. 4.II F and H).

A particularly striking observation was the DCL immunoreactivity inmitotic cells, in e.g. the ventricular zone and epithelial wall(examples in FIG. 4.II C-F, H, J-N), while also DCL positive, mitoticcells were found in the neuroepithelium of the neural tube (FIG. 4.II D)and the intermediate zone of the cortical neuroepithelium (FIG. 4.II F),generally with a more isolated occurrence and at lower frequencies. Inaddition to the DCL staining pattern of the epithelia in the samesection, clear immunopositive doublets were observed (FIG. 4.II D andF). Also mitotic cells in specific stages of the cell cycle could berecognized (FIG. 4.II J-N), with intense immunoreactivity betweenchromosomes and even immunopositive centrosome-like structures (FIG.4.II L) clearly visible.

Taken together, the data clearly showed that presence of DCL mRNAexpression precedes that of DCX starting already from ED8, and DCLprotein from ED9 onwards. Highest expression of DCL mRNA and protein wasfound at ED12 and ED14, respectively, while, contrary to DCX, alsotranscript and protein expression of DCL was found on Western blots fromadult brain. Protein distribution during early development is not onlydifferent from that of DCX in time, but also in location, i.e., DCL wasfound in the ventricular zone and cortical plate rather than thecortical plate alone (FIG. 4.II C, F-H). Most strikingly, DCLimmunoreactivity was regularly found in mitotic cells of theneuroepithelium and sometimes in the intermediate zone.

Example 5 DCL is Endogenously Expressed in Neuroblastoma Cells

To investigate a possible role for DCL in neuronal proliferation, theendogenous DCL expression in several neuronal cell lines was analysed. ADCL immunoreactive band of approximately 40 kDa was observed in 4different neuroblastoma cell lines, that was absent in any of thenon-neuroblastoma cell lines studied (FIG. 5.I A), indicatingspecificity for DCL expression in cells with a neuroblast-likephenotype. Screening of other non-neuroblastoma cell lines includingPC12 cells, failed to identify any DCL positive cell-line (data notshown). In the neuroblastoma cell line N1E-115, the 40 kDaimmunoreactive doublet co-migrated with the doublet resulting fromoverexpressing DCL (FIG. 5.I B). This 40 kDa band could not be explainedby the presence of DCX in N115 cells since both RT-PCR experiments andWestern blot analysis failed to detect DCX signals using DCX-specificprimers and antibodies (data not shown). The upper band of the 40 kDaDCL doublet therefore most likely represents a phospho-isoform of DCL, anotion that was confirmed by the disappearance of the upper band of boththe endogenous as well as overexpressed DCL when the cell lysates wereincubated with phosphatase. This further demonstrates that DCL, similarto DCX, is a phosphoprotein, at least in neuronal cell lines.

Example 6 DCL Affects Microtubule Architecture and Organization inN1E-115 Cells

To study function and subcellular localization of DCL,immunocytochemical experiments using confocal microscopy followingmanipulation of DCL expression using small interference (si) RNAtechnology in N1E-115 neuroblastoma cells in interphase was performed.To establish siRNA take up by N115 cells, anti-DCL synthetic siRNAmolecules were labelled with Cy-5 and their presence or absence wasmonitored in N115 cells by fluorescent microscopy. These studiesindicated the presence of anti-DCL siRNA in approximately 95% of allN115 cells (data not shown). Three different siRNA molecules against DCLwere constructed: siDCL-1, 2 and 3. Western blot analysis indicated thatsiDCL-1 failed to knock-down DCL protein (FIG. 5.II lane 1), a findingthat might be explained by the lack of TT di-nucleotides at the 3′-endin this antisense strand. siDCL-1 was subsequently used as a negativecontrol for the effects of the siRNA procedure. Compared withnon-treated cells and siDCL-1, transfection of siDCL-2 and si-DCL-3molecules lead to a knockdown of respectively 80% and 90% as determinedfrom Western blot analysis (FIG. 5.II lane 2 and 3). Subsequentimmunocytochemical analysis of siRNA treated N115 cells using anti-DCLKand α-tubulin or γ-tubulin antibodies revealed profound effects on thearchitecture of the microtubule cytoskeleton of cells in interphase. Innon-treated cells, anti-DCL staining was typically punctate and presentthroughout the remainder of the soma (see FIG. 5.III A). In contrast toDCX, which appears selectively located in the periphery of the soma andeven at the extremities of neuronal processes (Friocourt et al., 2003;Schaar et al., 2004), DCL immunoreactivity was less intense at theperiphery of the cell soma (FIG. 5.III A and C), and often displayedincreased intensity near one, or two sides of the nucleus (FIG. 5.III A,C, D and F), suggesting that DCL is concentrated particularly along thecytoskeleton surrounding the centrosome. This subcellular location wasconfirmed by co-staining with the centrosome marker γ-tubulin (see FIG.5.IV A-C).

In agreement with the Western blot analysis, transfection of siDCL-1 didnot alter the endogenous DCL immunocytochemical staining pattern. DCLknock-down induced by siDCL-3, however, induced a nearly completedisappearance of the anti-DCLK staining (see FIG. 5.III G), stronglyindicating that the anti-DCLK antibody recognizes DCL in N115 cells in ahighly specific manner. Strikingly, in 40% of the cells transfected withsiDCL-2 and in 80% of cells transfected with siDCL-3, the cytoskeletonwas disrupted, as was apparent from the altered, more dispersedα-tubulin staining pattern and irregular organization. N115 cellstransfected with siDCL-2 and 3 but with a normal cytoskeleton, alsoshowed more anti-DCLK staining than cells with an aberrant cytoskeleton.This further supports a causal relation between effective DCL knock-downand subsequent abnormalities in microtubule stability. Compared to thenormal microtubule cytoskeleton in non-treated cells, the abnormalpattern after DCL knockdown is characterized by bundles of microtubuleswith a more condensed and less dispersed structure, clearly exhibitingless side-branches (FIG. 5.III H and I). This indicated a role for DCLin branching and stabilization of the microtubule cytoskeleton.

Since DCL protein distribution was found in higher concentrations aroundthe centrosomes, DCL knockdown may affect centrosome protein complex andsubsequently nuclear positioning, cytoskeletal connectivity and(re-)organization. To address this issue, DCL knockdown was performed incombination with γ-tubulin staining (see FIG. 5.IV D-I). In agreementwith the euploid nature of N115 cells, multiple centrosomes per cellwere observed. However, despite efficient knock-down of DCL, no apparentchange was seen in the number or structure of centrosomes, indicatingthat DCL is not a key factor in the structural organization ofcentrosomes.

Example 7 DCL is Essential for Mitotic Spindle Formation inNeuroblastoma Cells

The presence of DCL in the ventricular zone (FIG. 4) is consistent witha role for DCL in neuronal proliferation and progenitor division.Dividing N1E-115 cells were therefore analysed using confocal microscopyfollowing DCL knockdown by siRNA. Strong DCL immunoreactivity wasobserved in all dividing N1E-115 cells during metaphase or earlyanaphase (see FIGS. 6A and D). DCL immunoreactivity largely colocalizedwith α-tubulin indicating an association with the mitotic spindles.However, an immunoreactive gradient was apparent for DCL in all cellsanalyzed, with low levels near the centrosome and high levels in themitotic spindles and near the kinetochore, suggesting a role for DCL inthe formation of mitotic spindles. Consistent with this are the dramaticeffects of DCL knockdown by siDCL-2 and siDCL-3, which are associatedwith a complete deformation and sometimes absence of the mitoticspindles (FIG. 6 G-L). This effect on the mitotic spindles was observedin 40% of all dividing cells (siDCL-2) and in all dividing cellstransfected with siDCL-3. Inefficient knockdown by siDCL-1 leaves DCLco-localization with mitotic spindles unaltered while the phenotypicappearance of mitotic spindles is similar to that of the non-treatedones mitotic spindles (FIG. 6 D-F). Thus, apparently, DCL is requiredfor the correct formation of the mitotic spindle of dividing neuroblastsor neural progenitors.

Example 8 DCL Overexpression Leads to Elongation of Mitotic Spindles

Gain-of-function was studied by overexpressing DCL in COS-1 cells thatnormally do not express this protein. Consistent with the above findingson endogenous DCL expression in dividing N115 cells, DCLimmunoreactivity co-localized with mitotic spindles in dividing COS-1cells (see FIG. 7). Two different phenotypes were observed: Firstly, in20% (n=126) of the analyzed dividing COS-1 cells, overexpression of DCLcolocalized with α-tubulin, similar to the endogenous DCL expressionpattern in dividing N1E-115 cells (FIG. 7 D-F), with DCL localized atthe kinetochore and in mitotic spindles. However, unlike N1E-115 cells,DCL is also found associated with the centrosomes and astral fibers.Secondly, in the majority of the dividing COS-1 cells (80%), comparisonof the precise mitotic stage of DCL expressing and vector-transfectedcells was hampered by the fact that all DCL expressing and dividingcells showed an abnormal phenotype with elongated mitotic spindles.

Most strikingly, half-spindles were observed indicating that DCLoverexpression affects centrosome segregation and spindle orientation(FIG. 7 A-C, G-I). In addition, the mitotic spindles appeared to be muchlonger and often thicker than the spindles from control cells (comparee.g. the spindle length of a non-transfected cell, FIG. 7B ref inset,with FIG. 7C). Notably, these DCL effects were associated with anabnormal DNA staining and distribution pattern, where the chromosomesare completely displaced and dispersed over the soma, a strikinglydifferent pattern from the normal orientation (FIG. 7B ref inset), thatis perpendicular with respect to the bipolar centrosome position (seereference length compared to FIG. 7C). Thus, overexpression of DCL inCOS-1 cells leads to spindle elongation and the formation ofhalfspindles, suggesting that DCL plays a crucial role in mitoticspindle form and length.

Example 9 Material and Methods

9.1 Cloning of the Murine DCL

The present inventors developed an antisense primer 1A: CTGGA ATTCTTACAC TGAGT CTCCT GAG (SEQ ID NO: 21) (EcoR1 site underlined)corresponding to the stop-codon region of the CARP-specific exon and asense primer 2S: GCAGG TTCTC ACTGA CATTA CCG (SEQ ID NO: 22)corresponding to exon 3 of the murine DCLK gene. In 30 cycles of PCR, a457 by fragment was amplified using mouse embryonic cDNA as a templateand polymerase PfuI (Stratagene). DNA sequence analysis confirmed theDNA sequence as being DCLK specific. Subsequently, a DCL cDNA encodingthe complete DCL protein was amplified usingCCAGGATCCACCATGTCGTTCGGCAGAGATATG (SEQ ID NO: 23) (Bamh1 siteunderlined) as a sense and 1A as an antisense primer, cut with Bamh1 andEcoR1 and subcloned in the expression plasmid pcDNA 3.1 (InVitrogen,Groningen, The Netherlands). A DCL-EGFP construct was generated bysubcloning a KpnI/EcoRV DCL fragment from pcDNA3.1.DCL in the SmaI/KpnIsite of pEGFP-C1 (Clontech; see also FIG. 1).

9.2 In situ hybridization

DCL mRNA includes exon 8 (FIG. 1), which is absent in most other DCLKtranscripts except for CARP. As CARP is expressed at very low levelsduring embryonic development, a 40-mer antisense oligonucleotide wasdeveloped (5′ -TTTGC TGTTA GATGC TTGCT TAGGA AATGG GAAAC CTTGA-3′) (SEQID NO: 24) complementary to an exon 8 specific sequence. As a negativecontrol the oligonucleotide 5′- TTTGATGTTA TATGC TTGAT TAGGA CATGG GACACCTGGA-3′ (SEQ ID NO: 25) which contains 6 mismatches (underlined), wasused. Both oligonucleotides were end-labelled with α-³³P dATP (NEN LifeScience Products, Hoofddorp, The Netherlands, 2000Ci/mmol, 10 mCi/ml)using terminal transferase according to the manufacturers instructions(Roche Molecular Biochemicals, Almere, The Netherlands). In situhybridization and visualization of the signals was performed asdescribed before (Meijer et al., 2000, Endocrinology 141, 2192-2199).

9.3 Antibodies

The generation of anti-DCL-antibodies has been described previously(Kruidering et al., 2001, supra). Mouse monoclonal anti-α-tubulin wasobtained from Sigma. Goat polyclonal anti-doublecortin (C-18) antibody,rhodamine-conjugated secondary antibodies and horseradishperoxidase-conjugated secondary antibodies were from Santa CruzBiotechnology, Inc.

9.4 Cell Culture and Treatments

All cell culture chemicals were obtained from Life Science Technologies,Inc. unless otherwise stated. All cells were maintained at 37° C., 5%CO₂. COS-1 cells were cultured in Dulbecco's modified Eagles medium(DMEM), supplemented with 100 units/ml penicillin, 100 μg/mlstreptomycin, and 10% Fetal Bovine Serum. NG108-15 and N115 cells werecultured in DMEM without sodium pyruvate, supplemented with 100 units/mlpenicillin, 100 μg/ml streptomycin, hybridoma (HAT) mix, and 10% FetalBovine Serum. For transient transfection experiments, cells werecultured on plates or coverslips coated with poly-L-lysine. Primarydissociated neurons from new born mice were cultured in F-12 Ham,Kaighn's modification (Sigma) medium supplemented with L-glutamine, 100units/ml penicillin, 100 μg/ml streptomycin, and 10% Fetal Bovine Serum.Primary neurons were isolated from the region of the hippocampus of aone day old mouse, that was incubated in a trypsin solution for 25minutes at 37° C. Subsequently, the cells were washed twice with culturemedium and plated on coverslips coated with poly-L-lysine. 24 Hourslater, the culture medium was replaced and supplemented with 7.5 μMcytosine-β-D-arabinoside (Sigma) to reduce the amount of glia cells. Thetransient transfection experiments were performed with Superfect(Qiagen, Valencia, Calif.) according to manufacturers instructions.Primary neurons were transfected four days after isolation.

9.5 siRNA Experiments

For siRNA experiments, the mouse neuroblastoma cell-line NIE-115 (ATCCnumber CRL-2263) was used. Synthetic RNA oligonucleotides 5′-CAAGA AGACGGCUCA CUCC-3′ (SEQ ID NO: 26) and 5′-GGAGU GAGCC GUCUU CUUG-3′ (SEQ IDNO: 27) (annealed siDCL-1), 5′- CAAGA AGACG GCUCA CUCCT T-3′ (SEQ ID NO:5) and 5′-GGAGU GAGCC GUCUU CUUGT T-3′ (SEQ ID NO: 6) (annealed siDCL-2)and 5′-GAAAG CCAAG AAGGU UCGAT T-3′ (SEQ ID NO: 9) and 5′-TCGAA CCUUCUUGGC UUUCT T-3′ (SEQ ID NO: 10) (annealed siDCL-3) in which the 3′thymidines are deoxynucleotides, were obtained from Eurogentec anddissolved in annealing-buffer (100 mM KAc, 30 mM Hepes pH7.5, 2 mM MgAc)to a final concentration of 100 pM. For the siRNA duplex formation,equal molar amounts of sense and antisense oligonucleotides were mixed,heated at 94° C. for 1 minute followed by incubation at 37° C. for 1hour. Per well a final concentration of 100 nM siRNA duplex was used.For gene silencing, 60 pmol siRNA duplex was dissolved in 50 μl opti-MEM(Life Technologies) and mixed by pipetting with 3 μp1 oligofectaminereagent (Invitrogen), dissolved in 12 μl opti-MEM. After 20 minutesincubation at room temperature, the volume was increased with 32 μlopti-MEM and the total mixture (100 μl) added to the cells (500 μl).After 48 hours, gene silencing was tested by Western blot analysis andimmunofluorescence.

9.6 Immunocytochemistry

Cells were cultured and transiently transfected as described above. Atthe indicated times, cells on coverslips were fixed with 80% aceton inwater for 5 minutes at room temperature. Cells were then rinsed twicewith phosphate-buffered saline (PBS), 0.05% Tween 20 and blocked for atleast 1 hour in blocking buffer: PBS, 0.05% Tween 20, 5% Normal GoatSerum (NGS, Sigma). Primary antibody was added for 1 hour at roomtemperature in blocking buffer, washed 3 times with PBS, 0.05% Tween 20and incubated with rhodamine-conjugated second antibodies for 30 minutesat room temperature in blocking buffer. Following another wash, thenuclei were stained with 0.2 μg/ml Hoechst 33258 for 5 minutes, washed 4times and analyzed. Images were obtained with an Olympus AX70fluorescent microscope coupled to a Sony 3CCD color video cameraoperated by Analysis® software (Soft Imaging System, Corp.). To map DCLprotein distribution, embryonic CD 1 mouse embryos of ED 9, 10 and 11were shortly washed in PBS and then fixed for 4 h inmethanol/acetone/water (40:40:20)(MAW, Franco et al, 2001) at roomtemperature and then stored in ethanol 70% for 2 weeks, before beingembedded in Paraplast Plus (Kendall, Tyco Healthcare, Mansfield, Mass.02048, USA) after which 6 μm thick sections were mounted on SuperfrostPlus slides (Menzel-Gläser). TBS was used as a washing buffer in allfollowing steps. After clearing in xylene and graded ethanol, sectionswere post-fixed in Bouin's fixative, prior to washing and blockage ofendogenous peroxidase activity by 15 min 0.1% hydrogen peroxidetreatment. To reduce aspecific binding, 1% milkpowder solution (Campina,The Netherlands) in PBS was applied for 30 min. The primary DCL antibodywas applied 1:50 in 0.25% gelatin/0.5% triton X-100 in TBS (Supermix)for 1 hour at room temperature and then overnight at 4° C. Secondaryantibody (biotinylated anti-rabbit, Amersham Life Sciences, 1:200)incubation was in Supermix for 1 h 30 min at room temperature, amplifiedwith avidin-biotin (ABC) Elite (Vector Laboratories, Burlingame),biotinylated tyramide (1:500) with 0.01% peroxide for 30 min followed byanother 45 min incubation with ABC. The last 2 washes were in 0.05 MTris HCl buffer (pH 7.6), which was also used to dissolvediaminobenzidine (DAB)(0.05 M). Sections were counterstained with cresylviolet and coverslipped with Entellan (Merck).

For comparison, also DCX protein distribution was mapped in adjacentsections, using the C-18 Doublecortin specific antibody (Santa CruzBiotechnology, South Cruz Calif., USA) at a 1:75 dilution. The sameprotocol was used as above, except for the blocking step in milkpowdersolution that was omitted and an biotinylated anti-goat as secondaryantibody.

9.7 Protein Extraction and Western Blotting

Mouse tissue and cells were solubilized with lysis buffer (20 mMtriethanolamine pH 7.5, 140 mM NaCl, 0.05% deoxycelate, 0.05% dodecylsodium sulfate, 0.05% Triton X100, supplemented with Complete™ EDTA-freeprotease inhibitor mixture (Roche Molecular Biochemicals) andcentrifuged at 16,000 g for 30 minutes. Supernatant was collected andprotein concentration determined using the Pierce method. Equal amountsof protein were separated by SDS-PAGE, transferred to immobilon-P PVDFmembranes (Millipore). Blots were blocked for 1 hour with blockingbuffer (Tris-buffered saline, 0.2% Tween 20 (TBST), 5% milk), incubatedwith primary antibodies in blocking buffer for 1 hour, washed 3 timeswith TBST, incubated with horseradish peroxidase-conjugated secondaryantibodies in blocking buffer for 30 minutes and washed 3 times withTBST. Antibody binding was detected by ECL (Amersham Pharmacia Biotech).

9.8 Phosphatase Treatment

DCL transfected and untransfected N1E-115 cells were solubilized withlysis buffer (50 mM Tris-HCl pH 9.3, 1 mM MgCl₂, 0.1 mM ZnCl₂, 1 mMspermidine supplemented with Complete™ EDTA-free protease inhibitormixture (Roche Molecular Biochemicals), centrifuged at 16,000 g for 30minutes. Supernatant was collected and protein concentration wasdetermined using the Pierce method. Each supernatant was divided in 3samples containing 50 μg of protein. One sample was untreated, thesecond incubated for 30 minutes at 37° C. without enzyme and the thirdwas incubated with 10 units of Calf Intestinal Alkaline Phophatase(Promega Bioscience, Inc.). The samples were analyzed by Westernblotting as described above.

9.9 Tubulin Polymerization Assay

DCL encoding cDNA was excised from the pcDNA3.1 expression construct andre-ligated into pET28 using BamH1 and EcoR1. The resulting DCLexpression construct was transfected into BL21 cells. A single colonywas grown in 500 ml LB to OD 0.7, at which point IPTG was added to afinal concentration of 0.4 mM. After three hours of induction, bacteriawere collected, washed with PBS and pelleted. Recombinant DCL proteinwas isolated by re-suspending the pellet and passing it through a Frenchpress after which it was purified using the Probond (Invitrogen) Ni²⁺affinity resin according to the manufacturer's instructions. PurifiedDCL was concentrated to 0.8 mg/ml using a Centricon 30 concentrationdevice. Tubulin polymerization assays were performed according toGleeson et al (Gleeson et al., 1999, supra) using the tubulinpolymerization assay kit (cat no BK006) from Cytoskeleton. Briefly, 1 mgtubulin was dissolved in 1.1 ml ice cold polymerization buffer accordingto the manufacturer's instructions and 100 μl of this was added to 10 μlDCL protein of various concentrations in a 96-wells microtiter plate.Subsequently, absorption at 340 nm was measured for 30′ in 30″ intervalsusing the HTS2000 (Biorad/Perkin Elmer).

Dcl and DCL SEQUENCES

SEQ ID NO: 1 ccacgcgtcc gcggagaacc gcatttcaat gaggaccagc tccagcgcatcagtgcacta gcggtcgcag cttccagacg ctcgtgctcc gcagccccag ccgcgcccagcccggcgagg acagctccag cagccggcca cagacaaccc agcctccacc cgcgaccggttccataagca agccagccat gtcgttcggc agagatatgg agttggagca ttttgatgagcgggacaagg cgcagaggta cagcaggggg tcccgtgtga atggcctgcc cagccccacacacagcgccc actgcagctt ctaccgcacc cgcaccctgc agacactcag ctccgagaagaaagccaaga aggttcgatt ctacagaaat ggtgaccgct acttcaaagg aattgtgtatgccatctccc cagaccgctt cagatctttc gaggccctgc tggctgattt gacccgaactctctcggata atgtgaattt gccccagggg gtgagaacca tctacaccat cgatggactcaagaagatct ccagcctgga ccagctggtg gaaggtgaaa gctatgtctg cggctccatcgagcccttta agaagctgga gtacaccaag aatgtgaacc ccaactggtc agtgaacgtcaagaccacct cagcctcccg cgcagtgtct tctttggcca ctgccaaggg tgggccttcggaggttcggg agaataagga tttcattcga cccaagctgg tcaccatcat cagaagtggggtgaagccac ggaaggctgt cagaatcctg ctgaacaaga agacggctca ctccttcgagcaggttctca ctgacattac cgacgctatc aagctggact ccggtgtggt gaagcgcctgtacactctgg atgggaagca ggtgatgtgc cttcaggact tttttggtga cgatgacatttttattgcat gtggaccaga gaagttccgt taccaggatg atttcttgct agatgaaagtgaatgtcgag tggtgaaatc aacttcttac accaaaatag catcagcgtc ccgcagaggcacaaccaaga gcccaggacc ttcccggaga agcaagtccc cagcctccac cagctcagttaatggaaccc ctggtagtca gctctctact ccacgctcgg gcaagtcacc aagtccatcacccaccagcc caggaagcct gcggaagcag agggacctgt accgccccct ctcgtcggatgatttggact caggagactc agtgtaagaa ttc SEQ ID NO: 2 gcacatccct gcactagtggccgcaaccga gacgccgcgc tccagcagct gctgccgccc agcccggccc cgccgccgccccccagccct gcagccccgc agccccggcc gcgcccagcc cggcgaggac agcaccaggaggcggccccc agcgcggcca caaagacccc cggcggcgtc tctccgcgga ccggtcctacttgaagtcca tcatgtcctt cggcagagac atggagctgg agcacttcga cgagcgggataaggcgcaga gatacagccg agggtcgcgg gtgaacggcc tgccgagccc gacgcacagcgcccactgca gcttctaccg cacccgcacg ctgcagacgc tcagctccga gaagaaggccaagaaagttc gtttctatcg aaacggagat cgatacttca aagggattgt gtatgccatctccccagacc ggttccgatc ttttgaggcc ctgctggctg atttgacccg aactctgtcggataacgtga atttgcccca gggagtgaga acaatctaca ccattgatgg gctcaagaagatttccagcc tggaccaact ggtggaagga gagagttatg tatgtggctc catagagcccttcaagaaac tggagtacac caagaatgtg aaccccaact ggtcggtgaa cgtcaagaccacctcggctt ctcgggcagt gtcttcactg gccactgcca aaggaagccc ttcagaggtgcgagagaata aggatttcat tcggcccaag ctggtcacca tcatcagaag tggcgtgaagccacggaaag ctgtcaggat tctgctgaac aagaaaacgg ctcattcctt tgagcaggtcctcaccgata tcaccgatgc catcaagctg gactcgggag tggtgaaacg cctgtacacgttggatggga aacaggtgat gtgccttcag gacttttttg gtgatgatga catttttattgcatgtggac cggagaagtt ccgttaccag gatgatttct tgctagatga aagtgaatgtcgagtggtaa agtccacttc ttacaccaaa atagcttcat catcccgcag gagcaccaccaagagcccag gaccgtccag gcgtagcaag tcccctgcct ccaccagctc agttaatggaacccctggta gtcagctctc tactccgcgc tcaggcaagt cgccaagccc atcacccaccagcccaggaa gcctgcggaa gcagagggac ctgtaccgcc ccctctcttc ggatgacttggattcagtag gagactcagt gtaaaagaaa SEQ ID NO: 3 Met Ser Phe Gly Arg AspMet Glu Leu Glu His Phe Asp Glu Arg Asp 1 5 10 15 Lys Ala Gln Arg TyrSer Arg Gly Ser Arg Val Asn Gly Leu Pro Ser 20 25 30 Pro Thr His Ser AlaHis Cys Ser Phe Tyr Arg Thr Arg Thr Leu Gln 35 40 45 Thr Leu Ser Ser GluLys Lys Ala Lys Lys Val Arg Phe Tyr Arg Asn 50 55 60 Gly Asp Arg Tyr PheLys Gly Ile Val Tyr Ala Ile Ser Pro Asp Arg 65 70 75 80 Phe Arg Ser PheGlu Ala Leu Leu Ala Asp Leu Thr Arg Thr Leu Ser 85 90 95 Asp Asn Val AsnLeu Pro Gln Gly Val Arg Thr Ile Tyr Thr Ile Asp 100 105 110 Gly Leu LysLys Ile Ser Ser Leu Asp Gln Leu Val Glu Gly Glu Ser 115 120 125 Tyr ValCys Gly Ser Ile Glu Pro Phe Lys Lys Leu Glu Tyr Thr Lys 130 135 140 AsnVal Asn Pro Asn Trp Ser Val Asn Val Lys Thr Thr Ser Ala Ser 145 150 155160 Arg Ala Val Ser Ser Leu Ala Thr Ala Lys Gly Gly Pro Ser Glu Val 165170 175 Arg Glu Asn Lys Asp Phe Ile Arg Pro Lys Leu Val Thr Ile Ile Arg180 185 190 Ser Gly Val Lys Pro Arg Lys Ala Val Arg Ile Leu Leu Asn LysLys 195 200 205 Thr Ala His Ser Phe Glu Gln Val Leu Thr Asp Ile Thr AspAla Ile 210 215 220 Lys Leu Asp Ser Gly Val Val Lys Arg Leu Tyr Thr LeuAsp Gly Lys 225 230 235 240 Gln Val Met Cys Leu Gln Asp Phe Phe Gly AspAsp Asp Ile Phe Ile 245 250 255 Ala Cys Gly Pro Glu Lys Phe Arg Tyr GlnAsp Asp Phe Leu Leu Asp 260 265 270 Glu Ser Glu Cys Arg Val Val Lys SerThr Ser Tyr Thr Lys Ile Ala 275 280 285 Ser Ala Ser Arg Arg Gly Thr ThrLys Ser Pro Gly Pro Ser Arg Arg 290 295 300 Ser Lys Ser Pro Ala Ser ThrSer Ser Val Asn Gly Thr Pro Gly Ser 305 310 315 320 Gln Leu Ser Thr ProArg Ser Gly Lys Ser Pro Ser Pro Ser Pro Thr 325 330 335 Ser Pro Gly SerLeu Arg Lys Gln Arg Asp Leu Tyr Arg Pro Leu Ser 340 345 350 Ser Asp AspLeu Asp Ser Gly Asp Ser Val 355 360 SEQ ID NO: 4 Met Ser Phe Gly Arg AspMet Glu Leu Glu His Phe Asp Glu Arg Asp 1 5 10 15 Lys Ala Gln Arg TyrSer Arg Gly Ser Arg Val Asn Gly Leu Pro Ser 20 25 30 Pro Thr His Ser AlaHis Cys Ser Phe Tyr Arg Thr Arg Thr Leu Gln 35 40 45 Thr Leu Ser Ser GluLys Lys Ala Lys Lys Val Arg Phe Tyr Arg Asn 50 55 60 Gly Asp Arg Tyr PheLys Gly Ile Val Tyr Ala Ile Ser Pro Asp Arg 65 70 75 80 Phe Arg Ser PheGlu Ala Leu Leu Ala Asp Leu Thr Arg Thr Leu Ser 85 90 95 Asp Asn Val AsnLeu Pro Gln Gly Val Arg Thr Ile Tyr Thr Ile Asp 100 105 110 Gly Leu LysLys Ile Ser Ser Leu Asp Gln Leu Val Glu Gly Glu Ser 115 120 125 Tyr ValCys Gly Ser Ile Glu Pro Phe Lys Lys Leu Glu Tyr Thr Lys 130 135 140 AsnVal Asn Pro Asn Trp Ser Val Asn Val Lys Thr Thr Ser Ala Ser 145 150 155160 Arg Ala Val Ser Ser Leu Ala Thr Ala Lys Gly Ser Pro Ser Glu Val 165170 175 Arg Glu Asn Lys Asp Phe Ile Arg Pro Lys Leu Val Thr Ile Ile Arg180 185 190 Ser Gly Val Lys Pro Arg Lys Ala Val Arg Ile Leu Leu Asn LysLys 195 200 205 Thr Ala His Ser Phe Glu Gln Val Leu Thr Asp Ile Thr AspAla Ile 210 215 220 Lys Leu Asp Ser Gly Val Val Lys Arg Leu Tyr Thr LeuAsp Gly Lys 225 230 235 240 Gln Val Met Cys Leu Gln Asp Phe Phe Gly AspAsp Asp Ile Phe Ile 245 250 255 Ala Cys Gly Pro Glu Lys Phe Arg Tyr GlnAsp Asp Phe Leu Leu Asp 260 265 270 Glu Ser Glu Cys Arg Val Val Lys SerThr Ser Tyr Thr Lys Ile Ala 275 280 285 Ser Ser Ser Arg Arg Ser Thr ThrLys Ser Pro Gly Pro Ser Arg Arg 290 295 300 Ser Lys Ser Pro Ala Ser ThrSer Ser Val Asn Gly Thr Pro Gly Ser 305 310 315 320 Gln Leu Ser Thr ProArg Ser Gly Lys Ser Pro Ser Pro Ser Pro Thr 325 330 335 Ser Pro Gly SerLeu Arg Lys Gln Arg Asp Leu Tyr Arg Pro Leu Ser 340 345 350 Ser Asp AspLeu Asp Ser Val Gly Asp Ser Val 355 360

1. A composition suitable for the treatment of neuroblastoma comprisinga pharmaceutically and/or physiologically acceptable carrier and atleast one antisense RNA or DNA oligonucleotide comprising one or moresequences selected from the group consisting of SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 19, and SEQ ID NO:
 20. 2. The composition accordingto claim 1, further comprising one or more targeting compounds, whereinsaid targeting compounds are capable of targeting neuroblastoma cells invivo or in vitro.
 3. The composition according to claim 2, wherein thetargeting compound is an immunoliposome or a monoclonal antibody.
 4. Thecomposition according to claim 1, wherein said at least one antisenseRNA oligonucleotide comprises 2′-O-methyl RNA or 2′-O-allyl RNA.
 5. Thecomposition according to claim 1, wherein said at least one antisenseRNA or DNA oligonucleotide comprises Peptide Nucleic Acids.
 6. Thecomposition according to claim 1, wherein said at least one antisenseRNA or DNA oligonucleotide comprises Locked Nucleic Acids.
 7. Thecomposition according to claim 1, wherein the amount of said at leastone antisense RNA or DNA oligonucleotide is between 0.05 and 5.0μmol/ml.
 8. A composition comprising a pharmaceutically and/orphysiologically acceptable carrier and a nucleic acid sequencecomprising SEQ ID NO: 2, or a complement thereof.
 9. A compositionsuitable for the treatment of neuroblastoma comprising at least oneantisense RNA or DNA oligonucleotide of 17-23 nucleotides comprising oneor more sequences selected from the group consisting of SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, and sequences derived thereof in which1, 2 or 3 nucleotides has been added, replaced or deleted.
 10. Thecomposition according to claim 9, further comprising a pharmaceuticallyand/or physiologically acceptable carrier.
 11. The composition accordingto claim 9, wherein at least one antisense RNA or DNA oligonucleotidecomprises Peptide Nucleic Acids.
 12. The composition according to claim9, wherein at least one antisense RNA or DNA oligonucleotide comprisesLocked Nucleic Acids.
 13. The composition according to claim 9, furthercomprising a targeting compound, which is capable of targetingneuroblastoma cells in vivo or in vitro.
 14. The composition accordingto claim 13, wherein the targeting compound is an immunoliposome or amonoclonal antibody.
 15. The composition according to claim 9, whereinsaid RNA oligonucleotide comprises 2′-O-methyl RNA or 2′-O-allyl RNA.16. The composition according to claim 9, wherein the amount of said atleast one antisense RNA or DNA oligonucleotide is between 0.05 and 5.0μmol/ml.